Activity modulator, medicinal agent comprising same, use of cd300a gene-deficient mouse, and anti-cd300a antibody

ABSTRACT

The present invention aims to provide: an immunostimulant useful for maintaining, enhancing or suppressing an immune function associated with CD300a activation signaling, or an immunomodulator as an immunosuppressant useful for suppressing the immune function; use of a CD300a gene-deficient mouse for pathology analysis and the like; an anti-CD300a antibody; and the like.

TECHNICAL FIELD

The present invention relates to an activity modulator for suppressingor promoting inhibitory signal transduction of a CD300a-expressingmyeloid cell, a medicament comprising it, use of a CD300a gene-deficientmouse, and an anti-CD300a antibody.

BACKGROUND ART

Invasion of a pathogen (bacterium, virus, parasite or the like) into ahost (human body or animal body) or generation of an endogenousinflammatory substance causes inflammatory reactions in which, forexample, temporary contraction of arteriolae occurs at the site ofinvasion of the pathogen or the site of generation of the inflammatorysubstance, and expansion and hyperemia then occur, leading to localslowness of blood flow at the site of invasion of the pathogen or thesite of generation of the inflammatory substance.

This causes adhesion of leukocytes to the vascular wall, and chemicalmediators released from various immunocytes then act on the leukocytesto cause them to pass through the vascular wall by amoeboid movement andto allow their migration. Known examples of the chemical mediatorsinclude histamine, serotonin and lymphokines. Mast cells, which produceand release histamine and serotonin, are a type of lymphocytes that playa central role in the inflammatory reaction. Similarly to mast cells,macrophages also produce and release chemical mediators such as TNF.

The leukocytes whose migration was induced by the inflammatory reactionare attracted by the pathogen or the like, and this causes elimination(clearance) of the pathogen from the body by humoral immunityaccompanied by antigen-antibody reaction and by cell-mediated immunityin which cytotoxic T cells and the like are involved, resulting inprevention of the spread of infection. Thus, the inflammatory reaction,and immune reactions that occur based on the inflammatory reaction, areextremely important for maintaining homeostasis of a living body.

On the other hand, the inflammatory reaction causes not only thebiological defense described above, but also adverse signs/symptoms suchas flare, fever, swelling, pain and dysfunction. Specific examples ofsuch symptoms include allergic diseases, and various types of acute andchronic inflammations. Also in autoimmune diseases, in which the absenceof immunological tolerance causes an autoimmune response, tissue injuryoccurs due to the inflammatory reaction.

That is, for prevention of a disease accompanied by the inflammatoryreaction, it is important to kill the pathogen that causes theinflammatory reaction using antibiotics (antimicrobial agents), or toadminister an agent that increases the immune function in the livingbody (immunostimulant) to eliminate the pathogen before an excessiveinflammatory reaction occurs.

On the other hand, known examples of methods for amelioration ortreatment of a disease accompanied by the inflammatory reaction includesuppression of inflammation by administration of an agent(anti-inflammatory agent (immunosuppressant)) that decreases excessivelyactivated immune function by, for example, suppression of release ofchemical mediators.

For example, Patent Document 1 discloses, as an immunostimulant, anactivating agent for the function of dendritic cells, which areantigen-presenting cells responsible for activation of variousimmunocytes. More specifically, the agent comprises as an effectivecomponent(s) at least one branched chain amino acid selected fromisoleucine, leucine and valine.

Patent Document 2 discloses, as an anti-inflammatory agent(immunosuppressant), an agent comprising the SPARC (Secreted proteinwhich is acidic and rich in cystein) peptide and a pharmaceuticalcarrier.

The following autoimmune diseases, allergic diseases and the like areknown.

Celiac disease, or coeliac disease, is an autoimmune disease and is aprogressive enteritis that is triggered by an immune reaction to gluten,which is a protein contained in wheat, barley, rye and the like. Theincidence of this disease in the United Kingdom, Europe and the UnitedStates is one or more per 300 individuals.

Gluten, which is a mixture of two proteins gliadin and glutenin, isfound in wheat, barley and rye. Gluten reacts with the small intestineand activates an immune system that attacks the fine small-intestinalepithelium, which is necessary for absorption of nutrients and vitamins,to cause injury.

When a patient with celiac disease takes a food or the like containinggluten, a gliadin-derived peptide, which cannot be digested by digestiveenzymes of human and is contained in wheat as a fraction of a plantprotein gluten, is deaminated by TG2 in the duodenal submucosal tissueto produce an antigen, which then causes production of autoantibodies.

Celiac disease occurs in genetically sensitive individuals having anyof: HLA-DQ2 (which is retained in about 90% of individuals), which isencoded by HLA-DQA1 and HLA-DQB1; HLA-DQ2 mutants; and HLA-DQ8.

Such individuals show induction of an immune response to peptidesinduced from water-insoluble proteins in flour, gluten, and relatedproteins in rye and barley, which immune response is limited toinappropriate HLA-DQ2 and/or DQ8 and mediated by CD4⁺ T cells.

This immune reaction triggers an attack of the autoimmune system on thesmall-intestinal epithelial tissue to cause inflammation and then injuryof villi and the like, leading to destruction of the epithelial cellsthemselves. As a result, nutrients cannot be absorbed from the smallintestine, and the patient suffers from malnutrition irrespective of thedietary intake and the like.

Ulcerative colitis, which is a representative inflammatory bowel disease(IBD), is a collective term for chronic diseases that cause inflammationof unknown origin mainly in the digestive tract, and is a chronicdisease in which inflammation occurs in the large intestine to formulcers.

Inflammatory bowel disease (IBD) is a collective term used fordescribing two gastrointestinal disorders (Crohn's disease (CD) andulcerative colitis (UC)) whose causes are unknown.

In Crohn's disease, a larger area is affected compared to ulcerativecolitis, and inflammation and ulcers are found in almost the whole areaof the digestive tract. Inflammatory bowel disease (IBD) occursworldwide, and as many as two million people are reported to havesuffered from Crohn's disease. The progression and prognosis of IBDwidely vary.

In inflammatory bowel disease (IBD), diarrhea and bloody stool continuefor a long period while the severity of symptoms changes with time. Thecause of the disease has not been elucidated, and, in a majorhypothesis, the continuous enteritis is thought to be caused by immunereactions to foods and enterobacteria due to disorder of immunologicaltolerance in the intestinal tract.

At present, there is no fundamental therapeutic method for the disease,and examples of therapies for inflammatory bowel disease includedietetic treatment; and use of an antidiarrheal (e.g., anticholinergic,loperamide or diphenoxylate), anti-inflammatory agent (e.g., steroiddrug such as aminosalicylic acid, sulfasalazine, mesalamine, olsalazine,balsalazide or prednisolone; or aminosalicylic acid) orimmunosuppressant (e.g., azathioprine, mercaptopurine or cyclosporin).

Surgical operations are required over a period of 10 years in 10% to 15%of the patients with IBD, and they have higher risk of occurrence ofintestinal cancer.

In Crohn's disease, bacteria are involved in the onset and theprogression of the disease, and intestinal inflammation in Crohn'sdisease is well-known for its frequent responsiveness to antibiotics andsusceptibility to bacterial fecal flow. Common intestinal microorganismsand novel pathogens have been suggested to have associations withCrohn's disease, based on direct detection or anti-microbial immuneresponses associated with the disease.

Moreover, in a number of genetically susceptible models of chroniccolitis, luminal microorganisms are indispensable cofactors for thedisease, and animals kept in a microbe-free environment do not developcolitis.

The combination of genetic factors, exogenous causes and the endogenousmicrobiota may contribute to the immune-mediated injury of theintestinal mucosa found in inflammatory bowel disease.

It is also known that development of ulcerative colitis is associatedwith polymorphisms in 3 gene regions, that is, the FCGR2A gene, whichencodes a receptor protein present on the surface of immunocytes;s8LC26A3, which encodes a transporter of chlorine ions and hydrogencarbonate ions; and a gene in the 13q12 region whose function is unknown(Non-patent Document 3).

However, in spite of abundant direct and indirect evidence on the roleof intestinal microorganisms in Crohn's disease, no pathogenic organismor antigen has been identified to contribute to the impairedimmunoregulation found in this disease. Tools useful for elucidation ofthe pathologies of the inflammatory bowel diseases (Crohn's disease andinflammatory enteritis), and medicaments for treatment and the like ofthese diseases have been demanded.

Atopic dermatitis is caused by entrance of an allergic substance(antigen) into the body followed by production of periostin due tostimulation by substances (interleukins 4 and 13) secreted fromactivated immunocytes, and then binding of the periostin to anotherprotein “integrin” on the surface of keratinocytes in the skin, to causeinflammation.

The binding of periostin to integrin causes production of otherinflammation-inducing substances, and the symptoms continue even in theabsence of the antigen, resulting in chronicity. It has been shown, byan experiment using mice, that inhibition of binding of periostin tointegrin using an inhibitor prevents occurrence of atopic dermatitis(Non-patent Document 4).

Although the major cause of atopic dermatitis has become evident,further elucidation of the pathology of atopic dermatitis, analysis ofassociation of atopic dermatitis with other inflammatory diseases, andmedicaments for atopic dermatitis that can be used in combination withthe above inhibitor, are demanded.

Bronchial asthma is a respiratory disease in which bronchialinflammation triggered by an allergic reaction or infection with abacterium or virus becomes chronic to thereby cause increased airwayhyperresponsiveness and reversible airway narrowing, leading to symptomssuch as attacks of wheezing, and cough. Further, bronchial asthma issaid to be caused by the combination of airway hyperresponsiveness,allergic diathesis and environment. Recurrent symptoms such as wheezing,apnea, chest tightness and cough occur especially at night or in theearly morning.

A number of cells and cellular components, especially mast cells,eosinophils, T-lymphocytes, macrophages, neutrophils and epithelialcells play roles in inflammation of the airway. Inflammation isassociated with plasma exudation, edema, smooth muscle enlargement,mucus plugging, and epithelial changes. Further, inflammation causesassociated increases in bronchial hyperresponsiveness to variousstimuli.

Inflammation of the airway induces atrophy of airway smooth muscle,microvascular rupture and bronchial hyperresponsiveness. As theresponsiveness of the airway increases, the symptoms become more severeand continuous, and daily variation of the pulmonary function increases.The mechanism of involvement of airway inflammation in the bronchialresponsiveness is unknown, and tools useful for elucidation of thepathology of asthma, and medicaments and the like have been demanded.

It is known that a group of receptor molecules called MAIR (MyeloidAssociated Ig like Receptors) are expressed on the cell membrane ofmyeloid (bone marrow) cells responsible for natural immunity (Non-patentDocument 1). Among these, MAIR-I, which is also known as CD300a (alsoreferred to as “LMIR1” or “CLM-8”), is expressed in macrophages, mastcells, granulocytes (neutrophils) and dendritic cells, and known to bean inhibitory receptor that associates with phosphatase via the ITIM(Immunoreceptor tyrosine-based inhibitory motif) sequence in theintracellular domain to transmit an inhibitory signal (Non-patentDocument 2). However, the ligand for this receptor is unknown, and thereceptor has been the so-called orphan receptor.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent Document 1] JP 2007-297379 A-   [Patent Document 2] JP 2011-516609 A

Non-Patent Documents

-   [Non-patent Document 1] Yotsumoto et al., J Exp Med 198 (2),    223-233, 2003-   [Non-patent Document 2] Okoshi Y et al., Int Immunol., 17, 65-72,    2005.-   [Non-patent Document 3] Asano K et al., Nature Genetics 41.    1325-1329 (2009)-   [Non-patent Document 4] Miho Masuoka et al., J Clin Invest. 2012;    doi: 10. 1172/JC158978

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The present invention aims to elucidate the regulatory mechanism ofactivation of the innate immune response via the inhibitory signaltransduction of CD300a, and to provide means for regulating the activityof CD300a-expressing cells involved in the inhibitory signaltransduction, means for treating or preventing diseases or diseasestates in which the activity is involved, and techniques and the likeassociated with these.

Means for Solving the Problems

In order to elucidate the ligand of CD300a and the function of CD300a,the present inventors intensively studied to discover the following.

(i) The ligand of CD300a is phosphatidyl serine (PS).(ii) Binding of PS to CD300a on mast cells and the like promotesinhibitory signal transduction via CD300a, and activation of the mastcells and the like are also suppressed thereby.(iii) Inhibition of binding of CD300a on mast cells and the like to PSby coexistence of a phosphatidyl serine-binding substance orCD300a-binding substance suppresses inhibitory signal transduction ofCD300a, and the active state of mast cells and the like is maintained.(iv) Through the suppression or maintenance of the active state,inflammatory infections, allergic diseases, autoimmune diseases and thelike can be treated.(v) CD300a gene-deficient mice can be a tool for performing pathologyanalysis of allergic diseases and autoimmune diseases, and screening ofeffective components of medicaments.

The present invention was carried out based on these discoveries, andprovides, for example, the activity modulators, medicaments, use of aCD300a gene-deficient mouse, anti-CD300a antibody, and the likedescribed in [1] to [22] below.

[1] An activity modulator for suppressing inhibitory signal transductionof a CD300a-expressing myeloid cell, the activity modulator comprising asubstance that inhibits binding of CD300a to phosphatidyl serine.[2] The activity modulator according to [1], wherein the substance thatinhibits binding of CD300a to phosphatidyl serine is a phosphatidylserine-binding substance.[3] The activity modulator according to [2], wherein the phosphatidylserine-binding substance is at least one selected from the groupconsisting of MFG-E8, MFG-E8 mutants, T cell immunoglobulin, solubleTIM-1, soluble TIM-4, soluble stabilin and soluble integrin αvβ3.[4] The activity modulator according to [1], wherein the substance thatinhibits binding of CD300a to phosphatidyl serine is a CD300a-bindingsubstance.[5] The activity modulator according to [4], wherein the CD300a-bindingsubstance is an anti-human CD300a antibody comprising: an H-chainvariable region having the amino acid sequence of SEQ ID NO:19 or anamino acid sequence that is the same as the amino acid sequence exceptthat 1, 2, 3, 4, or 5 amino acid(s) is/are substituted, added, insertedand/or deleted; and an L-chain variable region having the amino acidsequence of SEQ ID NO:20 or an amino acid sequence that is the same asthe amino acid sequence except that 1, 2, 3, 4, or 5 amino acid(s)is/are substituted, added, inserted and/or deleted.[6] The activity modulator according to [4], wherein the CD300a-bindingsubstance is an anti-mouse CD300a antibody comprising: an H-chainvariable region having the amino acid sequence of SEQ ID NO:17 or anamino acid sequence that is the same as the amino acid sequence exceptthat 1, 2, 3, 4, or 5 amino acid(s) is/are substituted, added, insertedand/or deleted; and an L-chain variable region having the amino acidsequence of SEQ ID NO:18 or an amino acid sequence that is the same asthe amino acid sequence except that 1, 2, 3, 4, or 5 amino acid(s)is/are substituted, added, inserted and/or deleted.[7] An activity modulator for promoting inhibitory signal transductionof a CD300a-expressing myeloid cell, the activity modulator comprising asubstance that promotes binding of CD300a to phosphatidyl serine.[8] The activity modulator according to [7], wherein the substance thatpromotes binding of CD300a to phosphatidyl serine is phosphatidylserine.[9] A medicament for treatment or prophylaxis of a disease or diseasestate in which inhibitory signal transduction of a CD300a-expressingmyeloid cell is involved, the medicament comprising the activitymodulator according to any of [1] to [8].[10] The medicament according to [9], wherein the disease or diseasestate in which inhibitory signal transduction of a CD300a-expressingmyeloid cell is involved is an inflammatory infection, allergic diseaseor autoimmune disease.[11] The medicament according to [10] for treatment or prophylaxis ofperitonitis or sepsis caused thereby, the medicament comprising theactivity modulator according to any of [1] to [6].[12] The medicament according to [10] for treatment of inflammatorybowel disease, the medicament comprising the activity modulatoraccording to any of [1] to [6].[13] The medicament according to [10] for treatment of celiac disease,the medicament comprising the activity modulator according to [7] or[8].[14] The medicament according to [10] for treatment of atopicdermatitis, the medicament comprising the activity modulator accordingto any of [1] to [6].[15] The medicament according to [10] for treatment of asthma, themedicament comprising the activity modulator according to any of [1] to[6].[16] Use of a CD300a gene-deficient mouse for carrying out pathologyanalysis of an allergic disease or autoimmune disease, or for screeningof a possible candidate substance for an effective component of atherapeutic agent or prophylactic agent for the disease.[17] The use according to [16], comprising use of the CD300agene-deficient mouse as a model mouse in which inflammatory boweldisease is hardly induced after administration of a substance thatinduces inflammatory bowel disease.[18] The use according to [16], comprising use of the CD300agene-deficient mouse as a model mouse that develops celiac disease afteradministration of a substance that induces celiac disease.[19] The use according to [16], comprising the step of: administering acandidate substance for a therapeutic agent for celiac disease to theCD300a gene-deficient mouse that developed celiac disease, anddetermining the presence or absence of a therapeutic effect; oradministering a candidate substance for a prophylactic agent for celiacdisease together with the substance that induces celiac disease to theCD300a gene-deficient mouse before development of celiac disease, anddetermining the presence or absence of a prophylactic effect.[20] The use according to [16], comprising use of the CD300agene-deficient mouse as a model mouse that hardly develops atopicdermatitis after administration of a substance that induces atopicdermatitis.[21] The use according to [16], comprising use of the CD300agene-deficient mouse as a model mouse that hardly develops asthma afteradministration of a substance that induces asthma.[22] An anti-human CD300a antibody comprising: an H-chain variableregion having the amino acid sequence of SEQ ID NO:19 or an amino acidsequence that is the same as the amino acid sequence except that 1, 2,3, 4, or 5 amino acid (s) is/are substituted, added, inserted and/ordeleted; and an L-chain variable region having the amino acid sequenceof SEQ ID NO:20 or an amino acid sequence that is the same as the aminoacid sequence except that 1, 2, 3, 4, or 5 amino acid(s) is/aresubstituted, added, inserted and/or deleted.[23] An anti-mouse CD300a antibody comprising: an H-chain variableregion having the amino acid sequence of SEQ ID NO:17 or an amino acidsequence that is the same as the amino acid sequence except that 1, 2,3, 4, or 5 amino acid(s) is/are substituted, added, inserted and/ordeleted; and an L-chain variable region having the amino acid sequenceof SEQ ID NO:18 or an amino acid sequence that is the same as the aminoacid sequence except that 1, 2, 3, 4, or 5 amino acid(s) is/aresubstituted, added, inserted and/or deleted.

As other aspects of the inventions described above, the followinginventions are provided.

Another aspect of the invention of [1] provides a method for suppressinginhibitory signal transduction of a CD300a-expressing myeloid cell,which method comprises inhibiting binding of CD300a to phosphatidylserine. This method is applicable either in vivo or ex vivo/in vitro,and, in cases of in vivo application, the species of organism may beeither human or non-human (e.g., mammal such as mouse).

Still another aspect of the invention of [1] provides use of a substancethat inhibits binding of CD300a to phosphatidyl serine in production ofan activity modulator for suppressing inhibitory signal transduction ofa CD300a-expressing myeloid cell.

As the substance that inhibits binding of D300a to phosphatidyl serinefor carrying out the method or use, the phosphatidyl serine-bindingsubstance recited in [2], preferably MFG-E8 or the like recited in [3]may be used. Similarly, as the substance that inhibits binding of CD300ato phosphatidyl serine for carrying out the method, the CD300a-bindingsubstance recited in [4], preferably the anti-human CD300a antibodycomprising an H-chain variable region and an L-chain variable regionhaving the prescribed amino acid sequences recited in [5], or theanti-mouse CD300a antibody comprising an H-chain variable region and anL-chain variable region having the prescribed amino acid sequencesrecited in [6], may be used.

Another aspect of the invention of [7] provides a method for promotinginhibitory signal transduction of a CD300a-expressing myeloid cell,which method comprises promotion of binding of CD300a to phosphatidylserine. This method is applicable either in vivo or ex vivo/in vitro,and, in cases of in vivo application, the species of organism may beeither human or non-human (e.g., mammal such as mouse).

Still another aspect of the invention of [7] provides use of a substancethat promotes binding of CD300a to phosphatidyl serine in production ofan activity modulator for promoting inhibitory signal transduction of aCD300a-expressing myeloid cell.

As the substance that promotes binding of CD300a to phosphatidyl serinefor carrying out the method or use, the phosphatidyl serine recited in[8] may be used.

Another aspect of the invention of [9] provides a method for treatmentor prophylaxis of a disease or disease state in which inhibitory signaltransduction of a CD300a-expressing myeloid cell is involved, whichmethod comprises inhibiting or promoting binding of CD300a tophosphatidyl serine, thereby suppressing or promoting inhibitory signaltransduction of a CD300a-expressing myeloid cell. This method isapplicable either in vivo or ex vivo/in vitro, and, in cases of in vivoapplication, the species of organism may be either human or non-human(e.g., mammal such as mouse).

Still another aspect of the invention of [9] provides use of an activitymodulator that inhibits or promotes binding of CD300a to phosphatidylserine to thereby suppress or promote inhibitory signal transduction ofa CD300a-expressing myeloid cell, in production of a medicament fortreatment or prophylaxis of a disease or disease state in whichinhibitory signal transduction of a CD300a-expressing myeloid cell isinvolved.

Examples of the disease or disease state in the method or use includeinflammatory infection, allergic disease, and autoimmune disease asrecited in [10], and specific examples of the disease or disease stateinclude the peritonitis or sepsis caused thereby, inflammatory boweldisease, celiac disease, atopic dermatitis, and asthma as recited in[11] to [15].

Another aspect of the invention of [16] provides a method for carryingout pathology analysis of an allergic disease or autoimmune disease, orfor screening of a possible candidate substance for an effectivecomponent of a therapeutic agent or prophylactic agent for the disease,by using a CD300a gene-deficient mouse. The CD300a gene-deficient mouseused in the method is a model mouse that develops inflammatory boweldisease, celiac disease, atopic dermatitis or asthma as recited in [17],[18], [20] or [21] after administration of a substance that induces eachallergic disease or autoimmune disease. The method is especiallypreferably a method for celiac disease, comprising the prescribed stepsrecited in [19].

Effect of the Invention

The present invention enables preparation of an activity modulator thatcan suppress or promote inhibitory signal transduction of aCD300a-expressing myeloid cell, and production of a medicament fortreatment or prophylaxis of a disease or disease state in whichinhibitory signal transduction of a CD300a-expressing myeloid cell isinvolved, which medicament comprises as an effective component theactivity modulator. Further, the present invention enables use of aCD300a gene-deficient mouse as a model mouse or the like, and productionof an anti-CD300a antibody having excellent neutralizing action.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of flow cytometry analysis obtained in Example1A.

FIG. 2A shows the results of flow cytometry analysis obtained in Example1B.

FIG. 2B shows the results of flow cytometry analysis obtained in Example1B.

FIG. 2C shows the results of flow cytometry analysis obtained in Example1C.

FIG. 2D shows the results of flow cytometry analysis obtained in Example1D.

FIG. 2E shows the results of flow cytometry analysis obtained in Example1D.

FIG. 2F shows the results of immunoblotting analysis obtained in Example1E.

FIG. 3A is a schematic diagram for illustrating the structure of theCD300a gene in the wild-type allele, the targeting vector used forpreparing a CD300a gene-deficient mouse, and the targeted allele.

FIG. 3B is a photograph taken after electrophoresis of PCR products fromthe wild-type allele and the mutant allele.

FIG. 3C shows the results of Western blotting using a wild-type mouseand a CD300a-deficient mouse.

FIG. 3D shows the results of flow cytometry analysis of a WT mouse and aCD300a gene-deficient mouse.

FIG. 4A shows the results of flow cytometry analysis obtained in Example2A.

FIG. 4B shows the results of analysis under a light microscope obtainedin Example 2C.

FIG. 4C shows the results of laser scanning confocal microscopy obtainedin Example 2C.

FIG. 4D shows the results obtained in Example 2C illustrating the ratioof the number of cells of NIH3T3 or each transfectant showingincorporation of a thymocyte in the cytoplasm.

FIG. 5A shows the results of flow cytometry analysis obtained in Example2B.

FIG. 5B shows the results of RT-PCR analysis obtained in Example 2B.

FIG. 6A shows the results of flow cytometry analysis obtained in Example3A.

FIG. 6B shows the results of analysis by the β-hexaminidase assayobtained in Example 2A.

FIG. 7A shows the results of flow cytometry analysis obtained in Example3B.

FIG. 7B shows the results obtained in Example 3C on the rates ofincrease in the amounts of various cytokines and chemokines released.

FIG. 7C shows a diagram showing the results obtained in Example 3E onthe rates of increase in the amounts of various cytokines and chemokinesreleased.

FIG. 7D shows the results of immunoblotting analysis obtained in Example3F.

FIG. 7E shows a diagram showing the results of immunoblotting analysisobtained in Example 3G.

FIG. 7F shows a diagram showing the rate of increase in TNF-α, obtainedin Example 3G.

FIG. 8 shows the results of flow cytometry analysis obtained in Example3D.

FIG. 9A shows the results of densitometric analysis obtained in Example4B.

FIG. 9B shows the results of densitometric analysis obtained in Example4B.

FIG. 10A shows a diagram shoveling the results of calculation of the CFUof aerobic bacteria, obtained in Example 4C.

FIG. 10B shows a diagram showing the numbers of neutrophils andmacrophages obtained in Example 4C after induction of CD300aneutrophils.

FIG. 11 shows a diagram showing the rate of the number of each type ofmacrophages that showed phagocytosis of E. coli.

FIG. 12A shows a diagram showing the results of flow cytometry analysisobtained in Example 4D.

FIG. 12B shows a diagram showing the rate of survival of each type ofmice, obtained in Example 4D.

FIG. 12C shows a diagram showing the bacterial clearance in theintestine in each type of mice in Example 4D, in terms of the bacterialCFU.

FIG. 12D shows the results of flow cytometry analysis obtained inExample 4E.

FIG. 12E shows a diagram showing the rate of survival of each group ofmice after administration of TX41 in Example 4F.

FIG. 12F shows a diagram showing the clearance in the intestine afteradministration of TX41 in Example 4G.

FIG. 12G shows a diagram showing the result of analysis of the change inthe number of neutrophils after administration of TX41 in Example 4G.

FIG. 13A shows a diagram illustrating the protocol for induction ofasthma with ovalbumin.

FIG. 13B shows the total cell number in the alveolar lavage fluid fromeach mouse on Day 25 after the beginning of induction of asthma inExample 5A.

FIG. 13C shows the ratio of eosinophils in the alveolar lavage fluidfrom each mouse on Day 25 after the beginning of induction of asthma inExample 5B.

FIG. 14 shows the serum IgE value in each mouse on Day 14 after thebeginning of induction of asthma in Example 5B.

FIG. 15 shows a diagram showing changes in the body weight with time dueto DSS-induced enteritis in each group of mice (Example 6A).

FIG. 16A shows a graph showing the length of the large intestine in eachtype of mice on Day 6 after the beginning of administration of DSS(Example 6B).

FIG. 16B shows a diagram showing sections of large intestines of a WTmouse and CD300a^(−/−) (Example 6C).

FIG. 17 shows a diagram for comparison of the degree of cell damage ineach type of mice on Day 6 of administration of DSS, based on theclinical score (Example 6D).

FIG. 18A shows a diagram showing the amount of each cytokine in thelarge intestine of each mouse on Day 9 of administration of DSS (Example6E).

FIG. 18B shows a diagram showing the number of each type of immunocyteson Day 0, Day 2, Day 4 and Day 6 after the beginning of administrationof DSS in each type of mice (Example 6F).

FIG. 19 shows a diagram showing the results of flow cytometry that wasperformed for identifying dendritic cells expressing CD300a (Example6G).

FIG. 20 shows a diagram showing the relative gene expression levels ofcytokines in the large intestine in each type of mice (Example 6H).

FIG. 21 shows a diagram showing the relative gene expression levels ofcytokines in CD4⁺ T cells (Example 6I).

FIG. 22 shows a diagram showing changes with time (daily changes) in thenumber of times of scratching behavior per 30 minutes in each type ofOVA-sensitized mouse (Example 7A).

FIG. 23 shows sections of the skin of each mouse at the end of the 3rdweek after sensitization with OVA (Example 7B).

FIG. 24 shows a diagram showing the result of toluidine blue staining ofa skin sample from each mouse at the end of the 3rd week aftersensitization with OVA (Example 70).

FIG. 25A shows a graph showing the number of cell layers in theepidermis in each group of mice at the end of the 3rd week aftersensitization with OVA (Example 7D).

FIG. 25B shows the numbers of eosinophils and mast cells that showedinfiltration into the dermis in skin samples of each group of mice atthe end of the 3rd week after sensitization with OVA (Example 7E).

FIG. 26 shows the result of Langerin immunostaining of a skin samplefrom each group of mice at the end of the 3rd week after sensitizationwith OVA (Example 7F).

FIG. 27 shows counterstaining of each sample in FIG. 26.

FIG. 28 shows a diagram showing a state where mast cells are interactingwith Langerin-positive skin cells (Example 7H).

FIG. 29 is a diagram showing a schedule of the test for confirming thetherapeutic effect of TX41.

FIG. 30 shows the total serum IgE level in WT mice after administrationof TX74 or TX41, as measured by the ELISA method.

FIG. 31 shows a diagram showing the number of times of scratchingbehavior in WT mice after administration of TX74 or TX41.

FIG. 32 shows a diagram showing the result of H&E staining of a skinsection of a WT mouse after administration of TX74 or TX41.

FIG. 33 shows a diagram showing a state in which a skin section of a WTmouse after administration of TX74 or TX41 was counterstained bytoluidine blue staining.

FIG. 34 shows a diagram showing changes in the body weight (BW) withtime monitored from the beginning of feeding with a normal diet orhigh-gluten diet.

FIG. 35 shows photographs each showing the result of histopathologicalanalysis of the intestine of a mouse at Week 20 after the beginning offeeding with a normal diet or high-gluten diet.

FIG. 36 shows a diagram showing the clinical score at Week 20 after thebeginning of feeding with a normal diet or high-gluten diet in eachgroup of mice.

FIG. 37 shows a diagram showing the number of intraepitheliallymphocytes per 100 intestinal epithelium cells at week 20 after thebeginning of feeding with a normal diet or high-gluten diet in eachgroup of mice.

FIG. 38 shows a diagram showing the amount of transglutaminase 2 (TG2)in a suspension of the jejunum at week 20 after the beginning of feedingwith a normal diet or high-gluten diet in each type of mice.

FIG. 39 shows a diagram showing the results of flow cytometry analysisof expression of CD11b and CD11c in cells of the lamina propria gatedfor the CD45⁺PI⁻ cell population.

FIG. 40 shows a diagram showing the frequency of each type ofimmunocytes in the jejunal lamina propria in WT mice and CD300agene-deficient mice kept with a normal diet or high-gluten diet.

FIG. 41 shows a diagram showing the gene expression levels of variouscytokines and chemokines in lamina propria (LP) macrophages in eachgroup of mice.

FIG. 42 shows a diagram showing the result of analysis of expression ofCD300a (MAIR-I) in each type of immunocytes by flow cytometry.

FIG. 43 shows a diagram showing the relative gene expression levels ofcytokines and chemokines in CD11b⁺ dendritic cells in the lamina propria(LP) in each type of mice.

FIG. 44 shows a diagram showing changes in the expression levels ofgliadin-induced cytokines caused by addition of a recombinant mouseMFG-E8 protein.

FIG. 45 shows a diagram showing the result of histopathological analysisof the large intestine of a Balb/c WT mouse or CD300a gene-deficientmouse fed with a normal diet or high-gluten diet.

FIG. 46 shows a diagram showing the anti-gliadin IgG and IgA antibodytiters in sera derived from Balb/c WT or CD300a gene-deficient mice.

FIG. 47 shows a diagram showing changes in the rate of change in thebody weight with time (weekly changes) in WT mice and CD300agene-deficient mice fed with a gluten-free diet.

FIG. 48 shows a diagram showing the expression levels of cytokines inCD11b⁺ dendritic cells and macrophages in the normal state in the laminapropria (LP) in WT mice and CD300a gene-deficient mice.

FIG. 49 shows a diagram showing the expression levels of cytokines inmacrophages in thioglycolate-induced peritoneal exudate cells afterstimulation with the gliadin peptide P31-43.

FIG. 50 is a diagram showing the expression levels of IL-6, IL-15,TNF-α, IFN-β, MCP1 and MCP5 in CD11b⁺ dendritic cells in the laminapropria.

FIG. 51 shows a diagram showing the expression levels of IL-6, TNF-α andIFN-β in lamina propria (LP) macrophages in B6 WT mice or CD300agene-deficient mice after stimulation with gliadin.

FIG. 52 shows a diagram showing expression of cytokines and chemokinesin lamina propria (LP) macrophages (stimulated with gliadin) derivedfrom microbiota-depleted WT mice or CD300a gene-deficient mice.

FIG. 53 shows a diagram showing the frequency of phosphatidylserine-expressing cells in isolated lamina propria (LP) macrophages.

FIG. 54 shows a diagram showing the gene expression levels of αv and β3integrin subunits in lamina propria (LP) macrophages in WT mice andCD300a gene-deficient mice.

FIG. 55 shows a diagram showing the gene expression levels ofphosphatidyl serine receptors (TIM-1, TIM-4, Stabiln-2, SR-PSOX, BAI1and Mer).

FIG. 56 shows the results of homology analysis of each of the H-chainand L-chain of TX41 and TX49.

DESCRIPTION OF EMBODIMENTS

The activity modulator of the present invention, a medicament comprisingit, use of a CD300a gene-deficient mouse, and an anti-CD300a antibody,are described below in detail. Literatures used for mentioningconventional knowledge or a known test method on the immune mechanism,CD300a or the like are listed in the end of Examples.

[Activity Modulator]

The activity modulator of the present invention includes those forsuppressing inhibitory signal transduction of a CD300a-expressingmyeloid cell, as well as those for promoting such inhibitory signaltransduction.

The “CD300a-expressing myeloid cell” herein includes a mast cell,macrophage, neutrophil, dendritic cell and the like. CD300a is acollective term for those expressed in mammals such as human and mouse,and the species of organism is not limited.

The “inhibitory signal transduction” is signal transduction that occursby association of the inhibitory receptor CD300a with phosphatase viathe ITIM (Immunoreceptor tyrosine-based inhibitory motif) sequence inthe intracellular domain.

In the following description, since the activity modulator forsuppressing inhibitory signal transduction of a CD300a-expressingmyeloid cell may have an action to activate an immune function as aresult, it is also referred to as “immunostimulant” in the presentinvention (for example, incases where it is used as an effectivecomponent of a medicament for an inflammatory infection). On the otherhand, since the activity modulator for promoting inhibitory signaltransduction of a CD300a-expressing myeloid cell may have an action tosuppress an immune function as a result, it is also referred to as“immunosuppressant” in the present invention (for example, in caseswhere it is used as an effective component of a medicament for celiacdisease).

<First Activity Modulator>

The first activity modulator by the present invention comprises acomponent having an action to suppress inhibitory signal transductionvia CD300a. In the present invention, as such a component, a substancethat inhibits binding of phosphatidyl serine to CD300a, that is, aphosphatidyl serine-binding substance or CD300a-binding substance may beused. The first activity modulator may contain either one of these, ormay contain both of these.

(Phosphatidyl Serine-Binding Substance)

The phosphatidyl serine-binding substance as a first activity modulatoris not limited as long as it binds to phosphatidyl serine (PS), which isa ligand of CD300a, to inhibit interaction (binding) between thephosphatidyl serine and CD300a expressed in a myeloid cell.

Specific examples of the phosphatidyl serine-binding substance includeMFG-E8 (Milk Fat Globular Protein EGF-8); T cell immunoglobulin; andsoluble proteins such as soluble TIM-1, soluble TIM-4, soluble stabilinand soluble integrin αvβ3. Among these, MFG-E8 is preferred.

The phosphatidyl serine-binding substance is not limited to nativeproteins such as MFG-E8, and may be one having an amino acid sequence inwhich one or several amino acids are deleted, substituted and/or added(mutant) (for example, “D89E MFG-E8” in Examples) as long as the bindingcapacity to phosphatidyl serine is not lost.

Such a mutant can be prepared by a known method such as site-directedmutagenesis or random mutagenesis.

The “soluble protein” described above means a protein prepared bymodifying a native protein, such as a membrane protein, insoluble to thelater-described diluent or body fluid by, for example, deleting ahydrophobic domain or adding a hydrophilic peptide by a known geneticrecombination technique such that the protein becomes soluble to thediluent or body fluid.

(CD300a-Binding Substance)

The CD300a-binding substance as a first activity modulator is notlimited as long as it binds to CD300a to inhibit interaction (binding)between the CD300a expressed in a myeloid cell and phosphatidyl serine.

Specific examples of the CD300a-binding substance include neutralizingantibodies against CD300a. The neutralizing antibody may be a singleparticular type of monoclonal antibody, or may be a combination of 2 ormore types of monoclonal antibodies (or polyclonal antibodies). Further,the neutralizing antibody may be either a full-length antibody or anantibody fragment (Fab fragment, F(ab′)₂ fragment or the like).

The neutralizing antibody can be prepared by a known method. In cases ofa monoclonal antibody, anti-CD300a monoclonal antibodies can begenerally prepared by, for example, a procedure comprising immunizationwith CD300a, preparation of hybridomas, screening, culturing, andrecovery. From the thus prepared antibodies, an appropriate monoclonalantibody that has a desired capacity (neutralizing action) to inhibitbinding of CD300a to phosphatidyl serine and can exert the action andeffect of the present invention may be selected.

(TX41, TX49, and Antibodies Similar to these)

TX41 is an anti-mouse CD300a monoclonal antibody (rat IgG2a), and TX49is an anti-human CD300a monoclonal antibody (mouse IgG1). Both of theseare monoclonal antibodies prepared and used in the later-describedExamples, and excellent in the function to suppress signal transductionby inhibition of binding of CD300a to phosphatidyl serine. Therefore,these are preferred as the CD300a-binding substance in the presentinvention. However, anti-CD300a antibodies that can be used in thepresent invention are not limited to TX41, TX49, and antibodies similarto these (having a variable region with an equivalent amino acidsequence).

The variable region in the H-chain of TX41 has the amino acid sequenceof SEQ ID NO:17; the variable region in the L-chain of TX41 has theamino acid sequence of SEQ ID NO:18; the variable region in the H-chainof TX49 has the amino acid sequence of SEQ ID NO:19; and the variableregion in the L-chain of TX49 has the amino acid sequence of SEQ IDNO:20. Each of these variable regions contains 3 complementaritydetermining regions (CDRs) and 4 framework regions. FIG. 56 shows theresults of analysis of homology between the amino acid sequences of thevariable regions of TX41 and TX49 (for each of the H-chain and L-chain).

The binding substance for mouse CD300a is preferably an antibody inwhich the variable region in the H-chain has the amino acid sequence ofSEQ ID NO:17, and the variable region in the L-chain has the amino acidsequence of SEQ ID NO:18, according to TX41.

The binding substance for human CD300a is preferably an antibody inwhich the variable region in the H-chain has the amino acid sequence ofSEQ ID NO:19, and the variable region in the L-chain has the amino acidsequence of SEQ ID NO:20, according to TX49.

Further, the binding substance for mouse CD300a may also be an antibodyin which the H-chain variable region has an amino acid sequence that isthe same as the amino acid sequence of SEQ ID NO:17 except that 1, 2, 3,4, or 5 amino acid(s) is/are substituted, added, inserted and/ordeleted, or an antibody in which the L-chain variable region has anamino acid sequence that is the same as the amino acid sequence of SEQID NO:18 except that 1, 2, 3, 4, or 5 amino acid(s) is/are substituted,added, inserted and/or deleted (one of the H-chain and the L-chain mayhave the above-described mutation(s), or both of these may have theabove-described mutation(s)).

Further, the binding substance for human CD300a may also be an antibodyin which the H-chain variable region has an amino acid sequence that isthe same as the amino acid sequence of SEQ ID NO:19 except that 1, 2, 3,4, or 5 amino acid (s) is/are substituted, added, inserted and/ordeleted, or an antibody in which the L-chain variable region has anamino acid sequence that is the same as the amino acid sequence of SEQID NO:20 except that 1, 2, 3, 4, or 5 amino acid(s) is/are substituted,added, inserted and/or deleted (one of the H-chain and the L-chain mayhave the above-described mutation(s), or both of these may have theabove-described mutation(s)).

The sites of such mutations are preferably not in the CDRs or vicinitiesthereof in the variable regions. Further, incases where an amino acid issubstituted, the substitution is preferably conservative amino acidsubstitution, in which substitution occurs between amino acids havingsimilar side-chain structures and/or chemical properties.

The form (amino acid sequence, amino acid length) of the constantregion, that is, the Fab region excluding the above-described variableregion, and the Fc region, of the anti-CD300a antibody may be designedas appropriate as long as the action and effect of the present inventionare not inhibited, since the form of the constant region hardly affectsthe binding capacity to CD300a, that is, the neutralizing action.

That is, the anti-CD300a antibody can be prepared as a fusion proteincomposed of the above prescribed amino acid sequence of the variableregion and a known amino acid sequence of the constant region.

For example, use of a human constant region for preparation of ananti-human CD300a antibody as a human chimeric antibody is one ofpreferred embodiments. Such an anti-CD300a antibody can be prepared by aknown method.

For example, by synthesizing a DNA encoding the above prescribed aminoacid sequence of the variable region and linking the synthesized DNA toa DNA encoding an amino acid sequence of the constant region andanother/other necessary DNA(s) (transcription factor(s) and/or thelike), an expression vector for an anti-CD300a antibody gene can beconstructed. By introducing this vector to a host cell and allowingexpression of the gene, the anti-CD300a antibody of interest can beproduced.

The above-mentioned TX41 and TX49, and antibodies similar to these canbe potentially used also for an object other than the action and effectof the present invention, i.e., the inhibition of inhibitory signaltransduction that occurs due to binding of phosphatidyl serine toCD300a.

Moreover, by suppressing expression of CD300a in myeloid cells in theaffected area using an siRNA designed based on a gene sequence of CD300a(available from DNA databases such as DDBJ/EMBL/GenBank=INSD),therapeutic effects for the various diseases described above can beobtained as in the cases where the CD300a gene is deleted or binding ofCD300a to phosphatidyl serine is inhibited. In other words, an siRNAagainst the CD300a gene can also be said to be the substance thatinhibits binding of CD300a to phosphatidyl serine in the presentinvention.

(Use of First Activity Modulator)

The first activity modulator of the present invention can be used forsuppressing inhibitory signal transduction of a CD300a-expressingmyeloid cell. In this case, the myeloid cell may be either a myeloidcell present in the body, or a myeloid cell separated from the body orcultured in vitro.

By maintaining or increasing activation signaling via CD300a of themyeloid cell by the above action, intercellular signal transduction viachemical mediators released from the myeloid cell is also maintained orincreased, and inflammation, allergic reaction and the like that arecaused by further intercellular signal transduction that occursthereafter can then be influenced. Therefore, the first activitymodulator can be used as an effective component (for example, as animmunostimulant) of the specific medicaments described later. Further,for example, the first activity modulator can also be used as aneffective component of an agent to be used as a comparative analysistool for comparative analysis performed after amelioration of thedisease state of asthma, atopic dermatitis, inflammatory bowel diseaseor sepsis in a laboratory animal.

Those skilled in the art can sufficiently presume that CD300a-bindingsubstances (neutralizing antibodies such as TX41 and TX49) andphosphatidyl serine-binding substances (MFG-E8 and the like) can betherapeutic agents for diseases that have been found to showamelioration of symptoms by deletion of the CD300a gene (that is, bycomplete prevention of binding of CD300a to phosphatidyl serine), suchas atopic dermatitis and the like described in Examples.

On the other hand, those skilled in the art can also sufficientlypresume that phosphatidyl serine can be a therapeutic agent for diseasesthat have been found to show development or exacerbation of symptoms bydeletion of the CD300a gene (that is, by complete prevention of bindingof CD300a to phosphatidyl serine), such as celiac disease described inExamples.

<Second Activity Modulator>

The second activity modulator contains a component having an action topromote inhibitory signal transduction via CD300a (that is, to suppressactivation signaling of CD300a). In the present invention, such acomponent may be a substance that promotes binding of phosphatidylserine to CD300a. The substance is especially phosphatidyl serine, whichis a ligand of CD300a.

Further, by performing screening using the CD300a gene-deficient miceprovided by another aspect of the present invention, agonists (lowmolecular compounds, antibodies and the like) for CD300a having the sameaction as phosphatidyl serine may be discovered, and such agonists canalso be used as substances that promote binding of phosphatidyl serineto CD300a.

(Phosphatidyl Serine)

Phosphatidyl serine (PS) is a ligand for CD300a expressed in myeloidcells, and interaction (binding) between PS and CD300a promotesinhibitory signaling of CD300a-expressing cells. For example, in mastcells, inflammatory reaction-associated activities that cause release ofchemical mediators such as histamine, cytokines and chemokines aresuppressed via this inhibitory signaling. PS is industrially produced,and can be easily obtained.

For CD300a-expressing myeloid cells placed in vitro (in a testenvironment), apoptotic cells presenting PS (it is known that PS ispresent inside the cell (in the cytoplasm-side layer of the lipidbilayer) in a normal cell, but presented outside the cell uponoccurrence of apoptosis) can also be a second activity modulator.Further, liposomes and the like having a PS-containing lipid membraneformed in the outside can be potentially used as second activitymodulators.

(Calcium Salt)

Since the interaction between PS and CD300a in mast cells requirescalcium ions, the second activity modulator preferably contains acalcium salt that generates a calcium ion by ionization (e.g., calciumchloride).

The content of the calcium salt in the second activity modulator may bedetermined appropriately in consideration of the calcium ionconcentration in the site of administration, the amount of PS containedin the second activity modulator, and the like.

(Use of Second Activity Modulator)

The second activity modulator of the present invention can be used forpromoting inhibitory signal transduction of a CD300a-expressing myeloidcell. In this case, the myeloid cell may be either a myeloid cellpresent in the body, or a myeloid cell separated from the body orcultured in vitro.

By suppressing activation signaling via CD300a of the myeloid cell bythe above action, intercellular signal transduction via chemicalmediators released from the myeloid cell is also suppressed, andinflammation, allergic reaction and the like that are caused by furtherintercellular signal transduction that occurs thereafter can then beinfluenced. Therefore, the second activity modulator can be used as aneffective component (for example, as an immunosuppressant) of thespecific medicaments described later. Further, for example, the secondactivity modulator can also be used as an effective component of anagent to be used as a comparative analysis tool for comparative analysisperformed after amelioration of celiac disease in a laboratory animal.

[Medicament]

The medicament (pharmaceutical composition) of the present inventioncontains the activity modulator as described above as an effectivecomponent (e.g., immunostimulant or immunosuppressant), and may furthercontain various pharmaceutically acceptable additives (e.g., a carrier),if necessary.

Such a medicament can be formulated for treatment or prophylaxis of adisease or symptom (especially inflammation reaction) in whichinhibitory signal transduction of a CD300a-expressing myeloid cell isinvolved, such as an inflammatory infection, allergic disease orautoimmune disease.

The “treatment” includes not only curing of a disease or symptom, butalso amelioration (alleviation) of a disease or symptom. The“prophylaxis” includes not only prevention of a disease or symptom inadvance, but also prevention of recurrence of a disease or symptom aftercuring of the disease or symptom.

More specifically, by blending the first activity modulator as aneffective component, a medicament or the like for treatment orprophylaxis of bacterial peritonitis or sepsis caused thereby,inflammatory bowel disease, atopic dermatitis or asthma can be prepared.

Further by blending the second activity modulator as an effectivecomponent, a medicament or the like for treatment or prophylaxis ofceliac disease can be prepared.

The site of administration of the medicament is not limited, and may bea site where excessive immune function (inflammation reaction) isoccurring, depending on the disease or disease state to which themedicament is applied. Examples of the site include intraperitoneal,intratracheal, subcutaneous, intradermal, and intraurogenital sites.

Since myeloid cells that express CD300a are usually present insubmucosal tissues and connective tissues in mammals, the medicament ispreferably directly administered to the submucosal tissue or connectivetissue at the above-described site, or in the vicinity thereof. Theadministration may be carried out by injection such as intravenousinjection, intraarterial injection, subcutaneous injection, intradermalinjection, intramuscular injection or intraperitoneal injection. Forexample, in cases of treatment or prophylaxis of an inflammatoryinfection (e.g., bacterial peritonitis), intraperitoneal injection ispreferred.

The dose per administration and the number of doses of the medicament(or the effective component contained therein) vary depending on theage, sex and body weight of the patient; symptoms; degree of thetherapeutic effect required; administration method; treatment period;type of the effective component; and the like; and may be appropriatelycontrolled. The number of doses is, for example, 1 to several doses perday.

In cases where the medicament contains as an effective component aphosphatidyl serine-binding substance as the first activity modulator,the medicament may be formulated such that the dose per administrationof the phosphatidyl serine-binding substance is usually 3 to 15 mg,preferably 5 to 10 mg per 1 kg of the human or animal subjected to theadministration.

In cases where the medicament contains as an effective component aCD300a-binding substance as the first activity modulator, the medicamentmay be formulated such that the dose per administration of theCD300a-binding substance is usually 50 to 150 mg, preferably 50 to 100mg per 1 kg of the human or animal subjected to the administration.

In cases where the medicament contains as an effective component asecond activity modulator, the medicament may be formulated such thatthe dose per administration of phosphatidyl serine is usually 3 to 150mg, preferably 5 to 100 mg per 1 kg of the human or animal subjected tothe administration in view of further increasing the immunosuppressiveeffect.

(Pharmaceutically Acceptable Carrier)

The medicament of the present invention may contain a pharmaceuticallyacceptable carrier, if necessary.

The pharmaceutically acceptable carrier is not limited as long as itdoes not deteriorate the purpose of the medicament, and examples of thecarrier include diluents such as aqueous diluents and nonaqueousdiluents; stabilizers/preservatives such as antioxidants (e.g.,sulfite); buffers such as phosphates; emulsifiers such as surfactants;coloring agents; thickeners; local anesthetics such as lidocaine;solubilizers such as glycols; isotonic agents such as sodium chlorideand glycerin; and other additives.

For example, in cases where the dosage form of the medicament of thepresent invention is an injection solution, the effective component ispreferably dissolved or dispersed in a diluent by blending the diluentsuch that a desired viscosity and desired concentrations of componentsare achieved.

Examples of such a diluent include aqueous diluents such asphysiological saline and commercially available distilled water forinjection; and nonaqueous diluents such as polyethylene glycol, andalcohols including ethanol.

The medicament whose dosage form is an injection solution may besterilized by filtration through a filter, or may be sterilized byblending a microbicide or the like, according to a conventional method.

In cases where the activity modulator is administered as an injectionsolution, it may be in the form of an injection solution to be preparedat the time of use. For example, a solid dosage form containing theactivity modulator may be prepared by freeze-drying or the like, and maythen be dissolved or dispersed in a diluent to prepare an injectionsolution at the time of administration.

<Medicament for Inflammatory Infection>

Inhibition of binding of phosphatidyl serine to CD300a-expressing cells(mast cells) enables maintenance or improvement of the activity of themast cells. This increases the number of neutrophils, and activatesattack of neutrophils to pathogens (bacteria, parasites and the like),thereby improving the function to suppress the growth of pathogens, andthe pathogen clearance function. Thus, by using the first activitymodulator (phosphatidyl serine-binding substance or CD300a-bindingsubstance) as an effective component (immunostimulant), a medicament foran inflammatory infection can be obtained.

<Medicament for Inflammatory Bowel Disease>

Inhibition of binding of phosphatidyl serine to CD300a-expressing cells(CD11b-positive dendritic cells in the large intestinal lamina propria)increases production, by CD4⁺ cells and the like, of IL-10, which isknown to suppress growth induction of inflammatory T cells. Thissuppresses activation of inflammatory T cells, and allows maintenance ofhomeostasis of the gut immune system. Thus, by using the first activitymodulator as an effective component, a medicament for inflammatory boweldisease can be obtained.

<Medicament for Atopic Dermatitis>

Inhibition of binding of phosphatidyl serine to CD300a-expressing cellscan suppress cellular infiltration of eosinophils, mast cells (which areknown to interact with skin Langerin-positive dendritic cells in thedermis to activate CD4-positive T cells) and monocytes; suppresshyperplasia of epidermis (fibroblasts); and decrease the total serum IgElevel. Thus, by using the first activity modulator as an effectivecomponent, a medicament for atopic dermatitis can be obtained.

<Medicament for Asthma>

Inhibition of binding of phosphatidyl serine to CD300a-expressing cellscan suppress eosinophilic airway inflammation, and decrease the totalserum IgE level. Thus, by using the first activity modulator as aneffective component, a medicament for asthma can be obtained.

<Medicament for Celiac Disease>

In cases where binding of phosphatidyl serine to CD300a-expressing cells(macrophages in the large intestinal lamina propria) is maintained,MyD88- and TRIF-mediated inhibitory gliadin signaling pathways playprotective roles in the progression of celiac disease. Thus, by usingthe second activity modulator as an effective component(immunosuppressant), a medicament for celiac disease can be obtained.

[Use of CD300a Gene-deficient Mouse] <Uses Related to Celiac Disease>

According to a discovery obtained in the present invention, induction ofceliac disease by administration of a gluten-derived gliadin peptide,which is known to be a substance that induces celiac disease, to CD300agene-deficient mice causes more severe symptoms of celiac disease thanadministration to wild-type mice.

That is, a CD300a gene-deficient mouse can be used as a model mouse thatdevelops celiac disease by administration of a substance that inducesceliac disease.

The model mouse of celiac disease in the present invention is a mouse inwhich the CD300a gene is inactivated and release of anti-inflammatorycytokines from mast cells and macrophages is derepressed.

The mouse shows symptoms of celiac disease (inflammation of the smallintestine) in cases where the mouse is fed from an early stage (from thelate middle age) with a substance that induces celiac disease. Thus, themouse is useful for elucidation of the cause of celiac disease, andplays an important role in development of a therapeutic agent for celiacdisease.

Symptoms of celiac disease can be confirmed by, for example, a knownmethod in which an inflammation-inducing substance is administered tothe mouse, and a section of the small-intestinal epithelium of the mouseis then observed under the microscope.

The celiac disease model mouse of the present invention can be used alsofor screening of therapeutic agents for celiac disease. In particular,since the mouse securely shows symptoms of celiac disease, the mouse isuseful for development of not only therapeutic agents to be appliedafter the onset of symptoms of celiac disease, but also prophylacticagents for celiac disease.

Further, the model mouse is also useful for evaluation of therapeuticagents for celiac disease that have already been demonstrated to betherapeutically effective.

For example, the model mouse is useful for studying the optimalconcentration of a therapeutic agent that has a therapeutic effectwithin a certain range of concentration but does not have the effect ata concentration lower than the range and shows toxicity at aconcentration higher than the range.

More specifically, the celiac disease model mice are divided into a testmouse group and a control mouse group, and a therapeutic agent to betested is administered to the test mouse group. By comparing sections ofthe small-intestinal epithelium between the groups, the effect of thetherapeutic agent can be evaluated.

Further, by giving the therapeutic agent to the test mouse group atdifferent concentrations, a therapeutically effective and optimalconcentration of the therapeutic agent can be studied.

That is, the CD300a gene-deficient mouse can be employed for uses inwhich a candidate substance for a therapeutic agent for celiac diseaseis administered to the CD300a gene-deficient mouse with celiac diseaseto see whether the agent is therapeutically effective or not; uses inwhich a candidate substance for a prophylactic agent for celiac diseaseis administered together with the substance that induces celiac diseaseto the CD300a gene-deficient mouse before development of celiac diseaseto see whether the agent is prophylactically effective or not; and usesfor analyzing the pathology of celiac disease.

The CD300a (MAIR-I) receptor is positioned upstream of the signaltransduction to suppress production of anti-inflammatory cytokines.Therefore, by administration of a CD300a (MAIR-I) agonist or signaltransducer that maintains or promotes the signal transduction tosuppress production of anti-inflammatory cytokines, symptoms of celiacdisease are expected to be ameliorated.

Thus, the CD300a gene-deficient mouse can also be preferably used inscreening for finding agonists; for finding signal transducers thatcomplement signal transduction downstream of the MAIR-I receptor, andsubstances that induce such signal transducers; and for finding genesinvolved in production of such signal transducers and the like.

This screening can be carried out by differential analysis between theCD300a gene-deficient mice and WT mice by DNA microarray analysis,two-dimensional protein electrophoresis or the like.

<Uses Related to Atopic Dermatitis>

Atopic dermatitis is hypersensitivity associated with allergic reaction,and accompanied by skin inflammation (eczema or the like).

Atopic dermatitis is caused by entrance of an allergic substance(antigen) into the body followed by production of periostin due tostimulation by substances (interleukins 4 and 13) secreted fromactivated immunocytes and then binding of the periostin to anotherprotein “integrin” on the surface of keratinocytes in the skin, to causeinflammation.

The binding of periostin to integrin causes production of otherinflammation-inducing substances, and the symptoms continue even in theabsence of the antigen, resulting in chronicity. It has been shown, byan experiment using mice, that inhibition of binding of periostin tointegrin prevents occurrence of atopic dermatitis (Document 31 listedbelow).

According to a discovery obtained in the present invention,administration of a substance that induces atopic dermatitis (miteantigen, ovalbumin or the like) to CD300a gene-deficient mice causesmilder symptoms of atopic dermatitis than administration to wild mice.

That is, the CD300a gene-deficient mouse can be used as a model mousethat hardly develops atopic dermatitis. Further, the CD300agene-deficient mouse can be used for analysis of signaling pathwaysinvolved in atopic dermatitis, pathology analysis of atopic dermatitis,and the like in relation to IL-4 and IL-13 production.

<Uses Related to Asthma>

Bronchial Asthma is a respiratory disease in which bronchialinflammation triggered by an allergic reaction or infection with abacterium or virus becomes chronic to cause increased airwayhyperresponsiveness and reversible airway narrowing, leading to attacksof wheezing, cough, and the like. Further, bronchial asthma is said tobe caused by the combination of airway hyperresponsiveness, allergicdiathesis and environment.

According to a discovery obtained in the present invention,administration of a substance that induces asthma (mite antigen,ovalbumin or the like) to CD300a gene-deficient mice to induce asthmacauses milder symptoms of asthma than administration to wild mice.

That is, the CD300a gene-deficient mouse can be used as a model mousethat hardly develops asthma after administration of a substance thatinduces asthma, and can also be used for elucidation of signalingpathways involved in asthma, pathology analysis of asthma, and the like.

<Uses Related to Inflammatory Bowel Disease>

According to a discovery obtained in the present invention, induction ofinflammatory bowel disease in CD300a gene-deficient mice by a knownmethod such as administration of an inflammation-inducing substance(e.g., DSS) causes milder symptoms of inflammatory bowel disease than inwild-type mice.

That is, the CD300a gene-deficient mouse can be used as a model mousethat hardly develops an inflammatory disease after administration of asubstance that induces inflammatory bowel disease, and can also be usedfor pathology analysis and the like of inflammatory bowel disease inrelation to the causative gene of the disease.

(Method for Preparing CD300a Gene-Deficient Mouse)

The CD300a gene-deficient mouse of the present invention is a mouse inwhich the CD300a gene on the chromosome is replaced by an inactiveCD300a gene and hence the function of CD300a protein is deficient.

The “inactive CD300a gene” means a gene that is incapable of expressingnormal CD300a protein due to, for example, partial deletion of theCD300a gene, insertion of another base sequence to the coding region ofthe CD300a gene, a point mutation(s) in the CD300a gene, or mutation ina regulatory region for expression of the CD300a gene. Examples of thedeficient CD300a gene include, but are not limited to, genes in which atleast one of the exons 1 to 6 contained in the CD300a gene is deleted.

The term “function of CD300a protein is deficient” means that at least apart of the function of CD300a protein involved in the inhibitory signaltransduction related to the present invention is lost (for example, thefunction of CD300a protein is partially lost by replacement of one ofthe alleles by an inactive form, as in a heterozygous knockout mouse),preferably means that the function is completely lost.

A common method for obtaining a CD300a gene-deficient mouse using a genecassette is as follows. However, the CD300a gene-deficient mouse in thepresent invention is not limited to those obtained by this method.

A targeting vector having a targeted allele (mutant allele) in which theexons of the wild-type allele of the CD300a gene are replaced by anantibiotic resistance gene (marker gene) is prepared. According to aconventional method, a chimeric mouse is obtained using the targetingvector and ES cells. The chimeric mouse is crossed with a wild-typemouse to obtain F1 mice (heterozygote (+/−)), and the F1 mice arecrossed with each other to obtain F2 mice.

Genomic DNA is extracted from the F2 mice, and genomic DNA of each mouseis subjected to PCR to investigate the presence/absence of the wild-typeallele and the mutant allele in the genomic DNA, to obtain an F2 mousehaving only the mutant allele (homozygote (−/−)). Further, in order toconfirm the absence of expression of CD300a, cells derived from themouse are subjected to confirmation by Western blotting using ananti-CD300a antibody, to provide a CD300a gene-deficient mouse.

EXAMPLES

The present invention is described below more concretely by way ofExamples. However, the present invention is not limited by the Examplesbelow.

1. Preparation Example (1) Preparation of CD300a-Fc Fusion Protein,MFG-E8 and D89E MFG-E8

(1-1) A CD300a fusion protein having the Fc region of human IgG(CD300a-Fc) was prepared as described in the Document 25 listed belowfrom a chimeric cDNA containing a cDNA of a gene encoding the wholeextracellular domain of mouse or human CD300a and a cDNA of a geneencoding human IgG1Fc. The fusion protein in which the extracellulardomain of CD300a is derived from mouse is referred to as “mouseCD300a-Fc”, and the fusion protein in which the extracellular domain ofCD300a is derived from human is referred to as “human CD300a-Fc”.(1-2) MFG-E8 was provided by Mr. Masato Tanaka (RCAI, Yokohama, Japan).(1-3) D89E MFG-E8 is a mutant of MFG-E8 obtained by site-directedmutagenesis in the RGD motif of MFG-E8 as described in the Document 4below.

In the obtained D89E MFG-E8, the 89th amino acid as counted from theN-terminus is substituted from aspartic acid to glutamic acid. MFG-E8(native) binds to both phosphatidyl serine (PS) and αvβ3 integrin tothereby cross-link apoptotic cells to phagocytes expressing αvβ3integrin (Document 4 listed below). On the other hand, D89E MFG-E8 doesnot bind to av33 integrin while it binds to phosphatidyl serine (PS).

2) Mice, and Cecal Ligation and Puncture (CLP

The knockout mice used in the Examples were prepared or provided asfollows.

(i) CD300a Gene-Deficient (Cd300a^(−/−)) Mouse

Using a bacterial artificial chromosome (BAC) system, the exons 1 to 6of the wild-type allele of the Cd300a gene were replaced by a neomycinresistance gene cassette (PGK-GB2-neo) to prepare a targeted allele(mutant allele) (FIG. 3A). Subsequently, a chimeric mouse was obtainedaccording to a conventional method, and the chimeric mouse was crossedwith a wild-type mouse to obtain F1 mice (heterozygote (+/−)). The F1mice were crossed with each other to obtain F2 mice.

In order to select CD300a gene-deficient (Cd300a^(−/−)) mice from the F2mice, genomic DNA was extracted from the tail of each F2 mouse, and thegenomic DNA was subjected to PCR to investigate the presence/absence ofthe WT allele and the mutant allele in the genomic DNA.

As shown in FIG. 3B, the PCR product corresponding to the NT allele andthe PCR product corresponding to the mutant allele are detected as aband of about 540 bp and a band of about 700 bp, respectively.

Further, in order to confirm the absence of expression of CD300a in theCD300a gene-deficient mice, cells derived from the CD300a gene-deficientmice were subjected to Western blot analysis using an anti-CD300aantibody. As shown in FIG. 3C, a wild-type mouse showed a band of about50 kDa derived from CD300a, but this band was not detected in a CD300agene-deficient mouse.

(ii) C57BL/6J-kit^(W-sh/W-sh) Mouse

C57BL/6J-kit^(W-sh/W-sh) mice (hereinafter referred to as“kit^(W-sh/W-sh) mice” or “mast cell-deficient mice”) were provided fromRIKEN BioResource Center (Tsukuba, Japan). These mice are known to showdeficiency of mast cells (Document 21 listed below), and to showincorporation of DNA by phagocytes without apoptotic DNA fragmentation,followed by degradation of DNA in the phagocytes (Document 12 listedbelow).

(iii) CAD-Deficient Mouse

The CAD (Caspase-activated DNase)-deficient mouse described in theDocument 12 listed below was used.

(v) In Vivo Removal of Macrophages and Neutrophils

According to description in the Document 30 listed below, clodronateliposomes and control PBS liposomes (Encapsula NanoSciences) wereprepared. Subsequently, at Hour 24 after CLP, 0.5 mL of the liposomeswere injected to the abdominal cavity of the mouse to removemacrophages.

Further, at Hour 24 after CLP, an anti-Gr-1 antibody was injected intothe abdominal cavity of the mouse to remove neutrophils.

All the operations of preparation using mice and the evaluation tests inthe Examples were carried out in accordance with the guideline preparedby Animal Care and Use Committee of Laboratory Animal Resource Center,University of Tsukuba.

(3) Antibodies

The manufacturer, or the method for preparation, of each antibody isshown in the table below.

TABLE 1 Manufacturer or Antibody name preparation method Control ratantibody BD Pharmingen Anti-CD11b (M1/70) BD Pharmingen antibodyAnti-F4/80 antibody BD Pharmingen Anti-c-Kit (2B8) antibody BDPharmingen Anti-Gr-1 (RB6) antibody BD Pharmingen Anti-FcεRI (MAR-1) BDPharmingen antibody Anti-TNF-α antibody BD Pharmingen Human CD300amonoclonal Prepared according to a antibody TX49 (mouse IgG1) Document(*) listed below Mouse CD300a monoclonal Prepared according to aantibody TX41 (rat IgG2a) Document (*) listed below Anti-MCP-1 antibodyBioLegend Anti-IL-13 antibody eBiosciences Anti-SHP-1 (C-19) UpstateBiotechnology antibody Anti-SHP-2 (C-18) Upstate Biotechnology antibody(*): Preparation was carried out by the method described in the Document1 below.

(4) Preparation of Cells

Cells were prepared as follows.

(i) Bone Marrow-derived Mast Cells (BMMCs)

In complete RPMI 1649 medium supplemented with a cell growth factor(SCF) (10 ng/mL), IL-3 (4 ng/mL) and fetal bovine serum (FBS) (10%)placed in a 10-cm dish, 2×10⁸ mouse bone marrow cells were cultured fornot less than 5 weeks to prepare bone marrow-derived mast cells (BMMCs).The BMMCs were subcultured every week with fresh medium. Flow cytometryanalysis showed that more than 95% of the prepared BMCs werec-Kit⁺FcεRI⁺ cells.

(ii) Bone Marrow-Derived Macrophages (BMMφ)

In complete RPMI 1649 medium supplemented with M-CSF (10 ng/mL) andfetal bovine serum (FBS) (10%) placed in a 10-cm dish, 2×10⁶ mouse bonemarrow cells were cultured for 1 week to prepare bone marrow-derivedmacrophages (BMMφ).

(iii) NIH3T3 Cell Transfectant

According to a conventional method, a pMX-neo retrovirus vector plasmidcontaining a cDNA of Flag-tagged CD300a or cDNA of Flag-tagged CD300dwas prepared.

NIH3T3 cells were transfected with the thus prepared plasmid, to obtaina transfectant that stably expresses CD300a or CD300d. The transfectantthat stably expresses CD300a and the transfectant that stably expressesCD300d obtained are referred to as “NIH3T3 transfectant (CD300a)” and“NIH3T3 transfectant (CD300d)”, respectively.

The NIH3T3 cell transfectant that stably expresses TIM-4 was provided byMr. T. Kitamura (University of Tokyo). This transfectant is referred toas “NIH3T3 transfectant (TIM-4)”.

[Method of Evaluation]

Conditions for the method of evaluation are described below.

In the survival test, the Kaplan-Meier log-rank test was used, and, inother evaluation tests, the unpaired Student's t test was used toperform statistical analysis. Statistical significance was assumed atP<0.05.

(5) Binding Assay Etc. (i) Binding Assay

Cells were stained for 30 minutes in phosphate buffer supplemented with2% FBS in the presence or absence of 1 mM CaCl₂ using CD300a or acontrol human IgG, and then washed twice with the same buffer, followedby incubation with the F(ab′)₂ fragment of an FITC-conjugated goatanti-human IgG. Subsequently, for staining with annexin V, the cellswere incubated in 10 mM HEPES-NaOH buffer supplemented with 140 mM NaCland 2.5 mM CaCl₂ together with annexin V for 15 minutes at roomtemperature.

(ii) Binding Inhibition Assay

Cells were preincubated with a monoclonal antibody against mouse CD300a(TX41), control isotype antibody or MFG-E8 for 30 minutes, and thenstained with CD300a-Fc as in the above binding assay. Further, in orderto analyze whether CD300a-Fc was bound to phospholipid or not, an assaywas carried out using a PIP Strip (manufactured by Echelon Biosciences)according to the manufacturer's instructions.

(6) Measurement of CFU (Colony Forming Unit) of Aerobic Bacteria

Serial dilutions of mouse peritoneal perfusate were plated, and thedilutions were cultured on plates containing brain-heart infusion (BHI)agar at 37° C. for 48 hours. Subsequently, the CFU of aerobic bacteriawas calculated by measuring the number of colonies in 1 mL of theperitoneal perfusate as described in the Document 27 listed below.

Example 1 Identification of CD300a Ligand Example 1A

In order to confirm expression of the mouse CD300a ligand inhematopoietic stem cell lines and tumor cell lines, the following testwas carried out.

The bone marrow-derived macrophages (BMMφ) obtained in “1. PreparationExample”, bone marrow-derived dendritic cells (BMDCs) or IL-3-dependenthematopoietic cell line cells (BaF/3 cells) (2×10⁵ cells per each typeof cells) were incubated in PBS (phosphate buffered saline) containingCD300a-Fc (1 μg) and calcium chloride (1 mM) at 20° C. for 30 minutes,and then stained using a buffer containing an FITC-conjugated anti-humanIgG antibody (0.1 μg) and propidium iodide (PI) (1 μg).

The stained cells of each type were subjected to analysis using a flowcytometer (FACSCalibur, manufactured by Becton Dickinson; model number,“E6133”).

Further, a control test was carried out in the same manner as in theabove test method except that a control human IgG (1 μg) was usedinstead of mouse CD300a-Fc.

The results of flow cytometry analysis for BMMφ, BMDC and BaF/3 cellsare shown in FIG. 1A, FIG. 1B and FIG. 1C, respectively (the results ofthe control test are shown as “Ctrl Ig”).

As shown in FIG. 1A to FIG. 1C, it was found that, in cases wherecalcium chloride is contained, mouse CD300a-Fc binds to PI⁻ cells (livecells) but does not bind to PI⁺ cells (dead cells). That is, the mouseCD300a ligand is suggested to be expressed in dead cells.

Example 1B

In order to test whether mouse CD300a-Fc binds to apoptotic cells, whichare a type of dead cells, the following test was carried out.

Thymocytes derived from a C57BL/6 mouse (wild-type mouse) were incubatedwith dexamethasone (manufactured by Sigma) (10 μM) in RPMI medium toprepare apoptotic thymocytes.

The obtained apoptotic cells (cell number, 2×10⁵) were incubated in amedium (PBS) containing CD300a-Fc (1 μg), APC-conjugated annexin V(manufactured by BD Pharmingen) (1 μl) and calcium chloride (1 mM) at20° C. for 30 minutes, and stained using a buffer containing anFITC-conjugated anti-human IgG antibody (0.1 μg) and propidium iodide(PI) (1 μg).

The stained cells of each type were subjected to analysis using a flowcytometer (FACSCalibur, manufactured by Becton Dickinson; model number,“E6133”) (results: FIG. 2A).

Further, the cells were subjected to flow cytometry analysis under thesame conditions as in the above test except that a medium supplementedwith no calcium chloride was used instead of the medium supplementedwith calcium chloride (results: FIG. 2B).

According to FIG. 2A, it can be seen that, in the presence of calciumchloride, mouse CD300a-Fc bound to thymocytes that are stained withannexin V but did not bind to thymocytes that are not stained withannexin V (annexin V⁻). That is, it is suggested that mouse CD300a-Fcbinds to apoptotic thymocytes.

On the other hand, it can be seen as shown in FIG. 2B that mouseCD300a-Fc did not bind to apoptotic thymocytes in the absence of calciumchloride.

From these test results, it can be understood that mouse CD300a binds toapoptotic cells dependently on calcium ions.

Example 1C

The apoptotic cells obtained in Example 1B (cell number, 2×10) wereincubated in a medium (PBS) supplemented with CD300a-Fc (1 μg),APC-conjugated annexin V (manufactured by BD Pharmingen) (1 μl), controlhuman IgG1 (1 μg) and calcium chloride (1 μM) at 20° C. for 30 minutes,and stained using a buffer containing an FITC-conjugated anti-human IgGantibody (0.1 μg) and propidium iodide (PI) (1 μg).

The stained cells of each type were subjected to analysis using a flowcytometer (FACSCalibur, manufactured by Becton Dickinson; model number,“E6133”) (results: FIG. 2C “HuIgG1”).

Further, the cells were subjected to flow cytometry analysis under thesame conditions as in the above test method except that “TX41”, which isa monoclonal antibody, or “MFG-E8”, which is a protein expressed in themacrophage, was used instead of the control human IgG1 (results: FIG. 2C“TX41” and “MFG-E8”).

As described above, the “TX41” used herein is an anti-mouse CD300amonoclonal antibody, and blocks binding of mouse CD300a to the ligand.

“MFG-E8” is known to bind to both phosphatidyl serine (PS) and αvβ3integrin to thereby cross-link apoptotic cells to phagocytes expressingαvβ3 integrin (Document 4 listed below).

As can be seen by comparison among the results of flow cytometry shownin FIG. 2C, mouse CD300a did not bind to apoptotic cells in the presenceof TX41 or MFG-E8.

From this viewpoint, it is suggested that mouse CD300a-Fc has bindingcapacity to PS (that is, the ligand of CD300a is PS).

Example 1D

In order to test whether or not human CD300a binds to apoptotic cellssimilarly to mouse CD300a, the following test was carried out.

First, Jurkat cells (human T-cell line) were suspended in RPMI 1640medium, and the resulting medium was irradiated with UV for 60 minutesto prepare apoptotic Jurkat cells.

Flow cytometry analysis was carried out under the same test conditionsas in Example 1A except that the “Jurkat cell-derived apoptotic cells”were used as apoptotic cells instead of the apoptotic cells derived fromwild-type mouse thymocytes, and “human CD300a-Fc” was used instead ofthe “mouse CD300a-Fc” (results: FIG. 2D).

Further, cytometry analysis was carried out under the same testconditions as in Example 1B except that the “Jurkat cell-derivedapoptotic cells” were used as apoptotic cells instead of the apoptoticcells derived from wild-type mouse thymocytes; “human CD300a-Fc” wasused instead of the “mouse CD300a-Fc” and “TX49” or “control human IgG1”was used instead of “TX41” (results: FIG. 2E “TX49” or “HuIgG1”). “TX49”is an anti-human CD300a antibody (monoclonal antibody), and blocksbinding of CD300a to the ligand.

As can be seen from FIG. 2D to FIG. 2E, human CD300a-Fc bound to annexinV⁺ cells but did not bind thereto in the presence of the anti-humanCD300a antibody.

That is, it is suggested that, similarly to mouse CD300a-Fc, humanCD300a also binds to apoptotic cells.

Example 1E

Liquids (test liquids) containing various phospholipids (PS, PC, PE)(100 pmol) were spotted on a membrane (PIP-strip (manufactured byEchelon Bioscience)) to allow adsorption of the phospholipids on themembrane.

Subsequently, the membrane was immersed in TBST buffer (pH 8.0)containing mouse CD300a-Fc (1.5 μg/mL), supplemented with calciumchloride (1 mM) and BSA, at 20° C. for 2 hours.

Thereafter, the membrane was washed 3 times with TBST buffer that doesnot contain mouse CD300a-Fc (pH 8.0) to remove CD300a-Fc unbound to thephospholipids on the membrane. Detection was then carried out using TBSTbuffer (pH 8.0) containing HRP-conjugated anti-human IgG (manufacturedby Jackson Immun), supplemented with BSA (results: FIG. 2F).

In FIG. 2F, “PE”, “PC” and “PS” indicate the positions wherephosphatidyl ethanolamine, phosphatidyl choline and phosphatidyl serinewere spotted on the membrane, respectively, and “Blank” indicates aposition where no phospholipid was spotted on the membrane.

According to FIG. 2F, CD300a bound to neither PE nor PC, and boundspecifically to PS.

From the results of Examples 1A to 1E, it can be understood that CD300abinds to phosphatidyl serine (PS) dependently on calcium ions (that is,the ligand of CD300a is PS)

Example 2 Functional Analysis of CD300a (2)

Some PS-binding receptors are known to be expressed in phagocytes and tobe involved in removal of apoptotic cells under physiological andpathological conditions (Documents 4 to 9 listed below).

PS is known to mediate the so-called “eat me” signal in phagocytes(macrophages and the like), which are cells expressing CD300a (Documents10 and 11 listed below). In view of this, the tests described in theExamples 2A to 2C below were carried out to test whether CD300a isinvolved in phagocytosis of apoptotic cells or not.

Example 2A

Thymocytes derived from a CAD-deficient mouse were treated in the samemanner as in Example 1B to prepare apoptotic thymocytes.

Subsequently, macrophages (thioglycolate-induced peritoneal macrophages)derived from a CD300a gene-deficient mouse (2×10⁵ cells) wereco-cultured with the apoptotic thymocytes derived from a CAD-deficientmouse at a ratio of 1:5 (macrophages:apoptotic thymocytes (cellnumbers)) in a 8-well Lab-TeK II chamber slide (manufactured by NalgeNunc) at 37° C. for 1 hour.

Subsequently, as described in the Documents 5 and 26 listed below, theco-cultured macrophages were washed with cold PBS and fixed with afixative containing paraformaldehyde (1%). The fixed macrophages wherethen subjected to TUNEL staining using a buffer containing FITC-labeleddUTP (manufactured by Roche).

Not less than 50 cells randomly selected from the stained macrophageswere analyzed using a laser scanning confocal microscope (“FV10i”,manufactured by Olympus Corporation; product number, 1B22358), and thenumber of TUNEL-positive cells (apoptotic cells) contained per onemacrophage cell was counted. The ratios of macrophages containingapoptotic cells in the numbers of 0 to 8 (phagocytosis rates) werecalculated as percentages with respect to the total number ofmacrophages (results: FIG. 4A “Cd300a^(−/−)”).

Further, the phagocytosis rates were measured under the same conditionsas in the above test except that macrophages derived from a wild-typemouse were used instead of the macrophages derived from a CD300agene-deficient mouse (results: FIG. 4A “WT”).

As shown in FIG. 4A, no evident difference in the phagocytosis rate wasfound between the case where the macrophages were derived from a CD300agene-deficient mouse and the case where the macrophages were derivedfrom a wild-type mouse.

Example 2B

In order to test whether mast cells express known PS receptors (TIM-1,TIM-4, stabilin 2 and integrin αvβ3), the following test was carriedout.

Bone marrow-derived mast cells (BMMCs) (cell number, 2×10⁵) wereincubated at 20° C. for 30 minutes in a medium (PBS) containing a PE(Phycoerythrin)-conjugated TIM-1 monoclonal antibody (0.1 μg),APC-conjugated TIM-4 monoclonal antibody (0.1 μg) and Alexa-conjugatedanti-mouse CD300a monoclonal antibody (TX41) (0.5 μg).

Subsequently, the stained cells were washed twice using PBS, andsubjected to analysis using a flow cytometer (FACSCalibur, manufacturedby Becton Dickinson; model number, “E6133”) (results: FIG. 5A “BMMCs”).

Further, flow cytometry analysis was carried out under the sameconditions as in the above test method except that peritonealmacrophages or BM-derived macrophages were used instead of the BMMCs(results: FIG. 5A “Peritoneal macrophages” and “BM derivedmacrophages”).

Further, using High Capacity cDNA Reverse Transcription Kit(manufactured by Applied Biosystems), cDNAs were prepared fromperitoneal macrophages and BMMCs. Using each prepared cDNA, theexpression levels of stabilin 2, BA1-1, αv integrin, Cd300a and β-actin(loading control) were analyzed by RT-PCR (results: FIG. 5B).

As can be seen from FIG. 5A and FIG. 5B, unlike the cases ofmacrophages, the mast cells expressed CD300a and αvβ3 integrin, butshowed only low levels of expression of TIM-1, TIM-4 and stabilin 2,which are PS receptors involved in phagocytosis.

Example 2C

The NIH3T3 transfectant (CD300a) (cell number, 6×10⁴) was co-culturedwith FITC-labeled cells (apoptotic thymocytes or thymocytes (livecells)) for 2 hours, and washed with PBS, followed by analysis under alight microscope (BZ-9000, manufactured by Keyence) (results: FIG. 4B).

Further, the cells obtained after the co-culture and washing were fixedwith a fixative containing paraformaldehyde, Vectashield (manufacturedby Vector Laboratories), and analyzed using a laser scanning confocalmicroscope (results: FIG. 4C). In FIG. 4C, green areas (indicated byarrows) indicate phagocytosed cells (apoptotic thymocytes or thymocytes(live cells)).

Further, analysis using a light microscope and a laser scanning confocalmicroscope was carried out under the same conditions as in the abovetest except that NIH3T3 untransfected cells (negative control) or“NIH3T3 transfectant (TIM-4)” cells (positive control) were used insteadof the NIH3T3 transfectant (CD300a).

In FIG. 4B and FIG. 4C, “NIH-3T3” and “NIH-3T3/Tim4” show images of“NIH3T3” (untransfected cells) and “NIH3T3 transfectant (TIM-4)”,respectively, which images were obtained using alight microscope and alaser scanning confocal microscope.

The images obtained with the laser scanning confocal microscope wereused to measure the ratios of the number of untransfected cells and thenumber of cells of each transfectant that incorporated apoptoticthymocytes into the cytoplasm (percentages with respect to the number ofco-cultured untransfected cells or to the number of co-cultured cells ofeach transfectant) (results: FIG. 4D “apoptotic”).

Further, similarly, the ratios of the number of NIH3T3 cells and thenumber of cells of each transfectant that incorporated thymocytes (livecells) into the cytoplasm (percentages with respect to the number ofco-cultured untransfected cells or to the number of co-cultured cells ofeach transfectant) were measured (results: FIG. 4D “Live”).

As shown in FIG. 4B, unlike NIH3T3, both of the above transfectantsadhered to apoptotic thymocytes. However, based on FIG. 4C and FIG. 4D,it can be seen that only the NIH3T3 transfectant (TIM-4) incorporatedapoptotic thymocytes to show phagocytosis.

Although data are not shown, the NIH3T3 transfectant (CD300a) did notshow phagocytosis of live cells (thymocytes), similarly to NIH3T3.

From the results of Examples 2A to 2C, it can be understood that CD300ais not involved in phagocytosis of apoptotic cells by macrophages.

Example 3 Functional Analysis of CD300a (2)

As shown in FIG. 5, mast cells express CD300a, but, unlike macrophages,the cells do not express TIM-1, TIM-4 and stabilin, which are PSreceptors.

PS is known to bind to these PS receptors directly or indirectly, andcontribution of these receptors to incorporation of apoptotic cells isknown (Document 13 listed below). In view of this, the tests describedbelow in the Examples 3A to 3C were carried out in order to test whetherCD300a also has such a function or not (whether or not there isfunctional overlap in incorporation of apoptotic cells).

Example 3A

In complete RPMI 1649 medium supplemented with a cell growth factor(SCF) (10 ng/mL), IL-3 (4 ng/mL) and fetal bovine serum (FBS) (10%)placed in a 10-cm dish, 2×10⁸ bone marrow cells (BM cells) derived froma CD300a gene-deficient mouse were cultured for 4 weeks to prepare bonemarrow-derived mast cells (BMMCs) of the CD300a gene-deficient mouse.The BMMCs were subcultured every week with fresh medium.

Subsequently, the obtained BMMCs were incubated in RPMI1649 mediumcontaining an FITC-conjugated anti-FcεRIα antibody (0.1 μg) and aPE-conjugated anti-c-Kit antibody (0.1 μg/mL) at 4° C. for 30 minutes,and analyzed by flow cytometry (results: FIG. 6A “CD300^(−/−)”).

Further, flow cytometry analysis was carried out under the same testconditions as in the above test except that BMMCs were prepared usingbone marrow cells derived from a wild-type mouse instead of the bonemarrow cells derived from a CD300a gene-deficient mouse (results: FIG.6A “UT”). Each number in FIG. 6A indicates the ratio of the cells shownin each box. Each test was repeated 3 times independently.

Using each type of BMMCs, a β-hexosaminidase release assay(degranulation assay) was carried out as follows (for detailedconditions, see the Document 29 below).

First, 1×10⁵ to 2×10⁵ BMMCs of each type in the logarithmic Growth phasewere cultured at 37° C. for one day and night in a 24-well plate coatedwith gelatin (manufactured by Sigma), and then incubated at 37° C. for 1hour in a medium that contains a biotin-conjugated mouseanti-trinitrophenol IgE (0.5 mg/mL) but does not contain a supplement.

Subsequently, streptavidin was added to the medium to causecross-linking between the biotin-conjugated mouse anti-trinitrophenolIgE molecules, and culture was performed at 37° C. for 45 minutes,followed by collecting the supernatant.

To the collected supernatant, a buffer (pH 4.5) containingp-nitrophenyl-N-acetyl-β-D-glucosamide (manufactured by Sigma), citricacid (0.4 M) and sodium phosphate (0.2 M) was added, and the resultingmixture was incubated at 37° C. for 3 hours to allow hydrolysis reactionof p-nitrophenyl-N-acetyl-β-D-glucosamide by released β-hexosaminidase.This reaction was stopped by adding 0.2 M glycine-NaOH (pH 10.7), andthe absorbance at a wavelength of 415 nm, which increases as hydrolysisof p-nitrophenyl-N-acetyl-β-D-glucosamide proceeds, was measured toquantify the amount of β-hexosaminidase released. The rate (%) ofincrease in the amount of β-hexosaminidase released with respect to theamount observed with untreated BMMCs of each type is shown in FIG. 6B.

FIG. 6B shows the ratio of BMMCs that released β-hexosaminidase. Asshown in FIG. 6A and FIG. 6B, no significant difference was found inexpression of FcεRIα and c-Kit (marker proteins for mast cells) and therate of increase in the amount of β-hexosaminidase released between thecase where the BMMCs were derived from a CD300a gene-deficient mouse andthe case where the BMMCs were derived from a wild-type mouse.

That is, it can be seen that differentiation from bone marrow cells anddegranulation mediated by FceRI are not influenced by CD300a.

Example 3B

BMMCs derived from a CD300a gene-deficient mouse and the apoptotic cellsobtained in Example 1B (BMMCs:apoptotic cells=10:1 (ratio in terms ofthe cell number)) were incubated in PBS containing calcium chloride (1mM), APC-conjugated annexin V (1 μl), CD300a-Fc (1 μg/mL) and MFG-E8 (5μg) at 20° C. for 30 minutes, and then stained using a buffer containingan FITC-conjugated anti-human IgG antibody (0.1 μg/mL) and propidiumiodide (PI) (1 μg).

The stained cells were subjected to analysis using a flow cytometer(FACSCalibur, manufactured by Becton Dickinson; model number, “E6133”)(results: FIG. 7A “MFG-E8”).

Further, flow cytometry analysis was carried out under the sameconditions as in the above test except that a control IgG was usedinstead of MFG-E8 (results: FIG. 7A “Ctrl Ig”).

From the results shown in FIG. 7A, it can be seen that CD300a-Fc boundto apoptotic cells (annexin V⁺) in the presence of the control IgG, butthat the binding was specifically inhibited in the presence of MFG-E8(PS-binding substance).

The concentrations of cytokines and chemokines in the supernatant ofthis sample mixture were quantified using ELISA kits manufactured by BDPharmingen (TNF-α and IL-6) and R&D Systems (MIP-2, MCP-1, IL-13 andMIP-1α). As a result, none of the cytokines and chemokines could bedetected.

Example 3C

In order to test whether or not stimulation by LPS (lipopolysaccharide)changes the amounts of cytokines released in the coexistence of BMMCsand apoptotic cells, the following test was carried out.

BMMCs derived from a CD300a gene-deficient mouse and apoptotic cells(BMMCs:apoptotic cells=10:1 (ratio in terms of the cell number)) wereincubated in RPMI containing LPS (1 μg/mL) for 4 hours, and thesupernatant of the medium was then collected.

Subsequently, the levels of cytokines and chemokines were measured 3times using ELISA kits manufactured by BD Pharmingen (TNF-α and IL-6)and R&D Systems (MIP-2, MCP-1, IL-13 and MIP-1α), and the rate ofincrease in the amount of each cytokine or chemokine released withrespect to the amount observed with the BMMCs that had not beensubjected to the above LPS treatment was calculated (results: FIG. 7B“Cd300a^(−/−)”).

Further, the rate of increase in the amount of each cytokine orchemokine was calculated under the same conditions as in the above testexcept that BMMCs derived from a wild-type mouse were used instead ofthe BMMCs derived from a CD300a gene-deficient mouse (results: FIG. 7B“WT”).

As shown in FIG. 7B, LPS increased the amounts of cytokines released inboth types of BMMCs. However, the BMMCs derived from a CD300agene-deficient mouse showed significantly larger increases in theamounts of TNF-α, IL-13 and MCP-1 than the BMMCs derived from awild-type mouse.

Example 3D

Further, the following test was carried out in order to test the ratesof increase in intracellular cytokines and chemokines in BMMCs.

BMMCs derived from a CD300a gene-deficient mouse and the apoptotic cellsobtained in Example 1B (BMMCs:apoptotic cells=10:1 (ratio in terms ofthe cell number)) were incubated in a medium (RPMI) containing LPS(lipopolysaccharide) (1 μg/mL) for 4 hours, and the BMMCs and apoptoticcells after the incubation were then incubated in a medium (FIX & PERM,manufactured by Invitrogen) supplemented with fluorescently labeledantibodies against various cytokines and chemokines and formaldehyde at4° C. for 20 minutes. The stained cells were subjected to analysis usinga flow cytometer (FACSCalibur, manufactured by Becton Dickinson; modelnumber, “E6133”) (results: FIG. 8, arrows (1)). Further, flow cytometryanalysis was carried out under the same conditions as in the above testexcept that a control antibody was used instead of the fluorescentlylabeled antibodies against various cytokines and chemokines (controltest (results: FIG. 8, arrows (2))).

Further, flow cytometry analysis was carried out under the sameconditions as in the above test except that BMMCs derived from awild-type mouse were used instead of the BMMCs derived from a CD300agene-deficient mouse (results: FIG. 8, arrows (3)). Further, flowcytometry analysis was carried out under the same conditions as in theabove test except that a control antibody was used instead of thefluorescently labeled antibodies against various cytokines andchemokines (control test (results: FIG. 8, arrows (4))).

Each graph in FIG. 8 shows the amount of increase in MFI (meanfluorescence intensity) observed for each cytokinin or chemokine in eachtype of BMMCs, relative to that of LPS-untreated BMMCs.

According to FIG. 8, in both types of BMMCs, the amounts of cytokininsand chemokines in the cytoplasm increased compared to the case where theLPS treatment was not carried out. In particular, it can be seen thatthe BMMCs derived from a CD300a gene-deficient mouse showedsignificantly larger increases in the amounts of TNF-α and the like inthe cytoplasm than the BMMCs derived from a wild-type mouse.

Example 3E

D89E MFG-E8 is a variant (mutant) of MFG-E8 and has a point mutation(D89E) in the RGD motif. D89E MFG-E8 binds to PS, but does not bind toαvβ3 integrin.

In view of this, the following test was carried out in order to testwhether or not the amounts of cytokines and chemokines released fromBMMCs change in the presence of D89E MFG-E8.

TNF-α, IL-13, MCP-1 and IL-6 were quantified in the same manner as inExample 3C except that LPS as well as D89E MFG-E8 (5 μg/mL) were addedto the medium containing BMMCs derived from a CD300a gene-deficientmouse and apoptotic cells (results: FIG. 7C “CD300a^(−/−)”).

Further, TNF-α, IL-13, MCP-1 and IL-6 were quantified under the sameconditions as in the above test except that BMMCs derived from awild-type mouse were used instead of the BMMCs derived from a CD300agene-deficient mouse (results: FIG. 7C “UT”).

As shown in FIG. 7C, in the presence of D89E MFG-E8, no significantdifference was found in the concentrations of cytokines between the casewhere the BMMCs were derived from a CD300a gene-deficient mouse and thecase where the BMMCs were derived from a wild-type mouse.

Example 3F

CD300a is known to have an immunoreceptor tyrosine-based inhibitorymotif (ITIM) in the intracellular domain, and to induce SHP-1 uponcross-linking by an anti-CD300a antibody (Document 14 listed below).

In view of this, the following test was carried out in order to testwhether CD300a interacts with SHP-1 or not. As in Test Example 3C, BMMCsderived from a CD300a gene-deficient mouse or wild-type mouse wereco-cultured with apoptotic cells in the presence of LPS for 4 hours, anda homogenate of the cells was subjected to immunoprecipitation with ananti-CD300a antibody (TX41).

Using the thus obtained immunoprecipitates, immunoblotting with ananti-SHP-1 antibody or an anti-CD300a antibody was carried out asdescribed in Document 14 listed below (results: FIG. 7D).

As can be seen from these results, the BMMCs responded to thestimulation with LPS to induce (recruit) SHP-1 when they wereco-cultured with apoptotic cells. However, CD300a did not recruit SHP-1in the presence of D89E MFG-E8.

That is, it is thought that induction (recruitment) of SHP-1 by CD300ain response to LPS stimulation requires binding of PS to CD300a.

Example 3G

In order to investigate whether SHP-1 is involved in CD300a-mediatedsignaling or not, first, Ptpn6 (SHP-1 gene) of BMMCs derived from awild-type mouse was knocked out with an siRNA to prepare SHP-1-deficient(Ptpn6-KD) wild-type mouse-derived BMMCs under the following conditions.Further, similarly, SHP-1-deficient (Ptpn6-KD) CD300a gene-deficientmouse-derived BMMCs were prepared from BMMCs derived from a CD300agene-deficient mouse.

With 1 mL of X-treme Gene siRNA transfection reagent (manufactured byRoche), 0.5 mM siRNA (SHP-1 siRNA) (siGENOME SMARTpool; ThermoScientificDharmacom) targeting the SHP-1 gene (Ptpn6 gene) in BMMCs was mixed, and5×10⁵ BMMCs derived from a CD300a gene-deficient mouse were transfectedtherewith as described in the Document 28 listed below, to prepare SHP-1knockdown BMMCs derived from a CD300a gene-deficient mouse (BMMCs(CD300a^(−/−)•Ptpn6-KD)).

Further, SHP-1 knockdown BMMCs derived from a wild-type mouse (BMMCs(WT•Ptpn6-KD)) were prepared under the same conditions as in the abovetest except that BMMCs derived from a wild-type mouse were used insteadof the BMMCs derived from a CD300a gene-deficient mouse.

Here, in order to confirm that the BMMCs of each type were transfectedwith the SHP-1 siRNA and that the expression level of SHP-1 wasdecreased, a lysate of BMMCs (CD300a^(−/−)•Ptpn6-KD) or BMMCs(WT•Ptpn6-KD) was subjected to immunoblotting analysis using ananti-SHP-1 antibody, anti-SHP-2 antibody or anti-8-actin antibody(results: FIG. 7E).

As can be seen from FIG. 7E, the BMMCs transfected with the SHP-1 siRNAshowed a decreased expression level of SHP-1. “Ctrl” shows results ofimmunoblotting analysis using BMMCs transfected with a control siRNAinstead of the SHP-1 siRNA.

Subsequently, BMMCs (CD300a^(−/−)•Ptpn6-KD) or BMMCs (WT•Ptpn6-KD), andthe apoptotic cells obtained in Example 1B (BMMCs:apoptotic cells=10:1(ratio in terms of the cell number)) were incubated in RPMI supplementedwith calcium chloride (1 mM) and LPS (lipopolysaccharide) (1 μg/mL) for4 hours, and the amount of TNF-α released was measured in the samemanner as in Example 3B (results: FIG. 7F).

As shown in FIG. 7F (left graph), the BMMCs derived from a CD300agene-deficient mouse produced a significantly larger amount of TNF-αthan the BMMCs derived from a wild-type mouse. On the other hand, asshown in FIG. 7F (right graph), in the cases where the BMMCs derivedfrom a wild-type mouse or a CD300a gene-deficient mouse were transfectedwith the SHP-1 siRNA, the amount of TNF-α released was almost the samebetween the BMMCs derived from a wild-type mouse and the BMMCs derivedfrom a CD300a gene-deficient mouse, and no significant difference wasfound between these.

These results suggest that binding of PS to CD300a causes CD300a toinduce SHP-1 and to thereby mediate signaling that causes suppression ofthe activity of BMMCs, resulting in suppression of secretion of TNF-α.

From the results of Example 3, it can be understood that the interactionbetween PS and CD300a inhibits production of inflammation-inducing(LPS-inducing) cytokines and chemokines from BMMCs, and that theinteraction recruits SHP-1, resulting in suppression of secretion ofTNF-α.

Example 4 Functional Analysis of CD300a (3)

TNF-α, IL-3 and MCP-1 produced by mast cells are chemoattractants forneutrophils, and known to play important roles in bacterial clearance inCLP peritonitis mice (Patent Documents 15 to 19 listed below).

In view of this, in order to study whether CD300a has a bacterialclearance function or not, the Examples 4A to 4H below were carried out.

Example 4A

A wild-type mouse was subjected to midline incision of 1 to 2 cm on thececum (ventral region), and the end portion was ligated. Afterperforming two times of puncture using a 27-gauge needle in the ligatedarea, the cecum was returned to the abdomen. Thereafter, 1 mL of sterilephysiological saline was subcutaneously injected for rehydration, andthe incision site was closed by suturing. Details of the procedure andconditions for the CLP are described in the Document 16 listed below.

Before performing the CLP and 4 hours after performing the CLP,peritoneal perfusate was collected. Subsequently, APC-conjugated annexinV (1 μg) and CD300-Fc (1 μg) were added to the peritoneal perfusate, andstaining was performed with an FITC-conjugated anti-human IgG and PI(propidium iodide), followed by performing analysis by flow cytometry(results: FIG. 12A).

As can be seen from the results shown in FIG. 12A, the site ofperitonitis was a site where a number of cells were undergoingapoptosis, as described in the Document 20 listed below.

That is, the immune regulation by mast cells in the site of peritonitisis suggested to be influenced by CD300a.

Example 4B

In order to test the relationship between CD300a and the immuneregulation by mast cells, proteome analysis was carried out as follows.

First, CLP was carried out in the same manner as in Example 4A using awild-type mouse and a mast cell-deficient mouse (kit^(W-sh/W-sh)).

Four hours after performing the CLP, peritoneal perfusate was collectedfrom each mouse, and the collected peritoneal perfusate was subjected toproteome analysis of cytokines and chemokines using Proteome ProfilerArray (manufactured by R&D Systems) according to the manufacturer'sinstructions.

FIG. 9A shows the results of densitometry analysis (proteome analysis)using the peritoneal perfusate from each of the wild-type mouse and themast cell-deficient mouse (kit^(W-sh/W-sh) mouse) (in FIG. 9A, “PC”indicates a positive control).

FIG. 9B shows the pixel densities of the signals for the chemokines andcytokines, which were obtained from the densitometry images shown inFIG. 9A.

As can be seen from the results shown in FIG. 9B, at Hour 4 after theCLP, the concentrations of chemokines were higher in the kit^(W-sh/W-sh)mouse than in the wild-type mouse. Similar results were obtained in 2replicates of the test.

Example 4C

In the same manner as in Example 4B, peritoneal perfusate was collectedfrom wild-type mice and mast cell-deficient mice (kit^(W-sh/W-sh) mice)(n=3 per each type of mice).

Subsequently, a dilution series of each peritoneal perfusate wasprepared, and the prepared serial dilutions of peritoneal perfusate wereplated to perform culture on plates containing brain-heart infusion(BHI) agar at 37° C. for 48 hours. Thereafter, the CFU of aerobicbacteria was calculated by measuring the number of colonies in 1 mL ofthe peritoneal perfusate as described in the Document 27 listed below(results: FIG. 10A).

Further, the numbers of neutrophils and macrophages in each peritonealperfusate were also counted. The results are shown in FIG. 10B as“neutrophil” and “macrophage”, respectively.

From a wild-type mouse and mast cell-deficient mouse (kit^(W-sh/W-sh)),BM-derived macrophages were prepared. These macrophages (cell number:1×10⁶) were co-cultured for 1 hour in a medium containingfluorescein-labeled E. coli placed in a 24-well plate, and the number ofeach type of macrophages that phagocytosed E. coli was counted by flowcytometry to calculate the ratio of phagocytosing macrophages (results:FIG. 11 “BM macrophage”).

A test was carried out under the same conditions as in the above testexcept that PEC macrophages derived from a wild-type mouse or mastcell-deficient mouse (kit^(W-sh/W-sh) mouse) were used instead of theBM-derived macrophages derived from a wild-type mouse or mastcell-deficient mouse (kit^(W-sh/W-sh) mouse), to calculate the ratio ofphagocytosing macrophages (results: FIG. 11 “PEC macrophage”).

As shown in FIG. 10A, it can be seen that, at Hour 4 after the CLP, themast cell-deficient mouse (kit^(W-sh/W-sh) mouse) showed a lowerintraperitoneal bacterial CFU and a larger number of neutrophils thanthe wild-type mouse. On the other hand, as shown in FIG. 10B and FIG.11, the number of macrophages and their phagocytosis were notsignificantly different between the genotypes.

Example 4D

In order to test whether CD300a is involved in induction (recruitment)of neutrophils or not, the following Example was carried out.

To mast cell-deficient mice (Kit^(W-sh/W-sh) mice), PBS buffercontaining BMMCs derived from a wild-type mouse (cell number, 1×10⁶) wasadministered by intraperitoneal injection (n=20). On Day 28 after theadministration, CLP was performed in the same manner as in Example 4A,and the survival rate of the mice was measured (results: FIG. 12B “WTBMMCs→Kit^(W-sh/W-sh)”).

Further, a test was carried out under the same conditions as in theabove test except that BMMCs derived from a CD300a gene-deficient mousewere used instead of the BMMCs derived from a wild-type mouse, and thesurvival rate of mice was measured (results: FIG. 12B “CD300aBMMCs→Kit^(W-sh)/Kit^(W-sh)”). “Kit^(W-sh)/Kit^(W-sh)” in FIG. 12Bindicates the survival rate of mast cell-deficient mice (Kit^(W-sh/W-sh)mice) subjected to CLP without administration of BMMCs.

According to FIG. 12B, the mast cell-deficient mice (Kit^(W-sh/W-sh)mice) subjected to administration of BMMCs derived from a wild-typemouse showed a higher survival rate even after the CLP, compared to thecase where administration of BMMCs was not carried out.

However, it can be seen that the mast cell-deficient mice(Kit^(W-sh/W-sh) mice) subjected to administration of BMMCs derived froma CD300a gene-deficient mouse showed a significantly higher survivalrate even after CLP, compared to the mice subjected to administration ofBMMCs derived from a wild-type mouse and the mice that had not beensubjected to administration of BMMCs (FIG. 12B).

Further, as a result of measuring the bacterial CFU in the same manneras in Example 4C using peritoneal perfusate of each type of mice at Hour4 after the CLP, the mast cell-deficient mice (Kit^(W-sh/W-sh) mice)subjected to administration of BMMCs derived from a CD300agene-deficient mouse showed a significantly higher bacterial clearancethan other mice (results: FIG. 12C).

Example 4E

In order to test whether or not the amount of TNF-α released increasesby intraperitoneal administration of BMMCs to a mast cell-deficientmouse (Kit^(W-sh/W-sh) mouse), the following Example was carried out.

Twenty four hours before CLP, a CFSE-labeled BMMC mixture (BMMCs derivedfrom a CD300a gene-deficient mouse:BMMCs derived from a wild-typemouse=1:1 (ratio in terms of the cell number)) was administered to mastcell-deficient mice (Kit^(W-sh/W-sh) mice) by intraperitoneal injection.

The mice after injection of the BMMC mixture was subjected to CLP in thesame manner as in Example 4A, and, at Hour 4 after the CLP, peritonealperfusate was collected. Each type of BMMCs contained in the peritonealperfusate were subjected to analysis using a flow cytometer(FACSCalibur, manufactured by Becton Dickinson; model number, “E6133”)(results: FIG. 12D).

As shown in FIG. 12D, it can be seen that, at Hour 4 after the CLP, theBMMCs derived from a CD300D gene-deficient mouse (CD300a⁻CFSE⁺ cells)showed production of a significantly larger amount of TNF-α than theBMMCs derived from a wild-type mouse (CD300a⁺CFSE⁺ cells).

Example 4F

In order to test the influence of administration of an anti-CD300amonoclonal antibody (TX41) on CD300a, the following Example was carriedout.

First, CLP was carried out in the same manner as in Example 4A exceptthat 500 μg of an anti-CD300a monoclonal antibody (TX41) (n=13) wasintraperitoneally injected to wild-type mice 1 hour or 18 hours beforethe CLP, and the survival rate of the mice was measured in the samemanner as in Example 4D (results: FIG. 12E “Antibody to CD300a”).

Further, a test was carried out under the same conditions as in theabove test except that an isotype control antibody (n=11) was used as acontrol instead of TX41 (n=13), and the survival rate of the mice wasmeasured (results: FIG. 12E “Control antibody”).

As shown in FIG. 12E, it was found that the survival time of wild typemice was longer in the cases where CLP was carried out 1 hour or 18hours after administration of TX41 by intraperitoneal injection,compared to the cases of administration of the control antibody.

Example 4G

From each mouse at Hour 4 after the CLP in Example 4F, peritonealperfusate was collected. The obtained peritoneal perfusate was treatedin the same manner as in Example 4C to measure the bacterial CFU and thenumber of neutrophils (control antibody: n=5 and anti-CD300a monoclonalantibody: n=5) (FIG. 12F and FIG. 12G, respectively).

The administration of TX41 by intraperitoneal injection did not causedamage to myeloid cells including mast cells in the mice.

As shown in FIG. 12F and FIG. 12G, the administration of TX41 towild-type mice by intraperitoneal injection 1 hour or 18 hours beforeCLP resulted in a significant increase in neutrophils and an increasedbacterial clearance in the abdominal cavity.

From the results of Example 4, it can be understood that inhibition ofthe interaction between PS and CD300a by TX41 or the like allowsprevention of sepsis induced by peritonitis.

Under physiological conditions, a number of cells undergo apoptosis. Inthis process, PS receptors play a central role in incorporation of theapoptotic cells, and are indispensable for preventing the progression ofautoimmune diseases (Document 22 listed below).

On the other hand, it is known that, under pathological conditions suchas microbial infection, cell death due to apoptosis remarkablyincreases, and this causes inflammation reaction by mast cells viareceptors (e.g., Toll-like receptors) against pathogen-associatedmolecular patterns (PAMPs) (Documents 15, 23 and 24 listed below).Further, mast cells are known to play important roles in immune reactionagainst pathogens.

Thus, from the results in the above Examples, it can be understood thatPS not only provides an incorporation signal for phagocytes via severalkinds of PS receptors, but also has an effect to effectively suppressinflammation reaction caused by mast cells via CD300a, as newlydiscovered in the present invention.

It can therefore be understood that phosphatidyl serine-bindingsubstances (e.g., MFG-E8) and CD300a-binding substances (e.g.,neutralizing antibodies against CD300a) inhibit the interaction betweenPS and CD300a in mast cells to thereby activate the mast cells ormaintain such an activity.

That is, it can be understood that these substance are useful aseffective components of immunostimulants used for prophylaxis of variousLPS-induced inflammatory infections (and sepsis caused thereby).

Further, since PS suppresses activation signaling of CD300a and hencesuppresses activation of mast cells, PS can be understood to be usefulas, for example, an effective component of immunosuppressants to be usedfor suppressing inflammation reaction in allergic diseases andautoimmune diseases (for example, for suppressing release of chemicalmediators such as histamine) to alleviate or treat symptoms of theallergic diseases and autoimmune diseases (i.e., to suppress excessiveimmune function).

<Asthma> (Materials and Methods) (Mice)

C57BL/6J mice were purchased from Clea Japan, Inc. CD300a gene-deficientmice (CD300a mice) were obtained by crossing Balb/c CD300agene-deficient mice prepared in the inventors' laboratory with thepurchased WT C57BL/6J mice for 12 generations, and then performingback-crossing. Male and female mice that were 8 to 10 weeks old at thebeginning of induction of asthma were used.

(OVA-Induced Asthma)

FIG. 13A shows the protocol for inducing asthma with ovalbumin. On Days0, 7 and 14 after the beginning of induction of asthma, a mixture of 100of ovalbumin (OVA, chicken egg albumin, manufactured by Sigma) and 100μL of aluminum hydroxide gel (ALUM, ALhydrogel 2%, manufactured byInvitrogen) was intraperitoneally injected to each mouse.

Further, on Days 21, 22 and 23 after the beginning of induction ofasthma, each mouse was subjected to inhalation of 10% ovalbumin preparedby dilution with PBS, for 30 minutes using an ultrasonic nebulizer(NE-U17, Omuron). On Day 25 after the beginning of induction of asthma,each mouse was subjected to bronchoalveolar lavage (BAL), and serum wascollected from each mouse.

Example 5A FIG. 13A, FIG. 13B (Bronchoalveolar Lavage BAL)

After subjecting each mouse to tracheotomy, washing was performed 3times with 1 mL of 2% FBS/PBS, followed by collecting the washing liquidand measuring the cell number. As shown in FIG. 13A and FIG. 13B, thecell number in the bronchoalveolar lavage fluid (BAL fluid) of eachmouse on Day 25 after the beginning of induction of asthma wassignificantly smaller in the CD300a gene-deficient mice than in the WTmice in terms of both the total cell number and the number ofeosinophils. This result indicates that CD300a exacerbated eosinophilicairway inflammation caused by ovalbumin (OVA), and that the CD300agene-deficient mice showed amelioration of symptoms of eosinophilicairway inflammation.

Example 5B Analysis of Cells in Bronchoalveolar Lavage Fluid FIG. 13C

With CD45.2-FITC, Siglec-F-PE, CD11b-APC Cy7, CD11c-PEC Cy7 andF4/80-Alexa (all of these were purchased; BD), 1×10⁶ cells in thecollected bronchoalveolar lavage fluid were stained, andCD45⁺SiglecF⁻CD11b⁺CD11c⁻F4/80⁻ was analyzed as the eosinophil fractionby flow cytometry (FACS).

FACS analysis of cells in the bronchoalveolar lavage fluid of the miceon Day 25 after the beginning of induction of asthma showed the ratio ofeosinophils among CD45-positive cells (CD45⁺SiglecF⁻CD11b⁺CD11c⁻F4/80⁻).The CD300a gene-deficient mice showed a significantly smaller ratio ofinfiltrating eosinophils in the BAL fluid than the WT mice.

Example 5C Measurement of Serum IgE Value FIG. 14

Serum IgE was measured by ELISA using a rat anti-mouse IgE (BD) and abiotinylated anti-mouse IgE (BD).

As shown in FIG. 14, the CD300a gene-deficient mice showed significantlylower serum IgE values during the period of sensitization to causeovalbumin-induced asthma.

Based on comparison of the serum IgE value (index of the degree ofallergy) among the OVA-induced asthma model mice on Disease Day 14, theCD300a gene-deficient mice showed significantly lower serum IgE valuesthan the WT mice.

It is thought that administration of an anti-CD300a antibody, whichsuppresses signal transduction of CD300a, causes significant suppressionof serum IgE in ovalbumin-induced asthma.

<Enteritis> (Materials and Methods) (Mice)

C57BL/6 mice (WT mice) were purchased from Clea. CD300a gene-deficientmice established from Balb/cA-derived ES cells in the inventors'laboratory were backcrossed with C57BL/6, and mice of the 12th or latergeneration were used.

The mice used for the experiment were kept under a SpecificPathogen-Free (SPF) environment in Laboratory Animal Resource Center,University of Tsukuba.

(Induction of Enteritis)

Female C57BL/6 mice of 10 to 12 weeks old were made to drink 2.5% (w/v)dextran sulfate sodium salt, reagent grade (DSS, MP Biomedicals,molecular weight (MW)=36000 to 50000) continuously for 8 days forinduction of enteritis.

(Antibodies)

BD biotinylated anti-CD300a (TX41) was prepared in the inventors'laboratory. Other antibodies, which are described below, were purchasedfrom Bioscience.

-   -   Purified anti-mouse TNF-α    -   Biotin-labeled anti-mouse TNF-α    -   Purified mouse IL-6    -   Biotin-labeled anti-mouse IL-6    -   Purified anti-mouse IL-10    -   Biotin-labeled anti-mouse IL-10    -   Allophycocyanin-Cy7(APC-Cy7)-labeled anti-mouse CD11b (M1/70)    -   Phycoerythrin-Cy7(PE-Cy7)-labeled anti-mouse CD11c (HL3)    -   Fluorescein isothiocyanate (FITC)-labeled anti-mouse CD4 (L3T4)

(Large Intestine Tissue Culture Liquid)

After removal of the large intestine from each mouse on Day 9 afteradministration of DSS or water, the large intestine was washed withphosphate buffer to remove feces. The large intestine was longitudinallyincised, and 5 3-mm tissue pieces were cut out from the portion 3 mmdistant from the anus, and the tissue pieces were subjected to 12 hoursof culture in 10% FBS RPMI 1640 (manufactured by GIBCO). The supernatantwas then collected to provide a large intestine tissue culture liquid.TNF-α, IL-6 and IL-10 contained in the culture liquid were measured bysandwich ELISA.

(Isolation of Cells of Large Intestinal Lamina Propria)

Mice were sacrificed, and the large intestine of each mouse wascollected. The large intestine was cut into 4 pieces with scissors, andthen washed with phosphate buffer to remove feces. The remaining tissuewas placed in 1 mM DTT/5 mM MEDTA/Hank's balanced salt solution(manufactured by Sigma), and incubated in an incubator (shaker type) at37° C. for 30 minutes.

Subsequently, the tissue was washed again with phosphate buffer toremove detached epithelium and contaminants. The remaining tissue wascut into small pieces with scissors, and placed in 1 mg/mL collagenasetype 3 (manufactured by Worthington)/0.1 mg/mL DNase (manufactured byWorthington)/5% Fetal bovine serum/Hanks's balanced salt solution, andincubated in an incubator (shaker type) at 37° C. for 2 hours to allowcomplete lysis.

The cell suspension was passed through 70-μm nylon, and thencentrifuged. The obtained cells were suspended in 40% Percoll, andoverlaid on 70% Percoll. After centrifugation, cells in the intermediatelayer were collected to be used as cells of the large intestinal laminapropria in flow cytometry.

(Isolation of CD11b⁺CD11c⁺ Cells and CD4⁺ T Cells)

The isolated cells of the large intestinal lamina propria were labeledwith APC-Cy7-labeled anti-CD11b (M1/70), PE-Cy7-labeled anti-CD11c (HL3)and FITC-labeled anti-mouse CD4 (L3T4). The labeled cells were sorted byFACSAria (BD) to provide CD11b⁺CD11c⁺ cells and CD4⁺ T cells,respectively.

(Measurement of mRNA Level)

The cells sorted by FACSAria were lysed with ISOGEN-LS (Nippon Gene).From the lysed cells, mRNA was extracted using an mRNA extraction kit,and cDNA was prepared using a reverse transcription kit. The preparedcDNA was mixed with primers (see SEQ ID NOs:1 to 16 in SEQUENCE LISTING)and Power Cyber Green (Applied Biosystems), and measurement of the mRNAlevels was carried out using 7500 Fast Real Time PCR System (AppliedBiosysytems).

Example 6A Body Weight Change Rate (FIG. 15)

UT mice and CD300a gene-deficient mice were made to drink 2.5% (w/v)dextran sodium sulfate (DSS) continuously for 8 days to induceenteritis, and the body weight of each mouse was measured every day.

The mice used were female mice of 10 to 12 weeks old, and the rate ofchange in the body weight was observed for 15 UT mice and 15 CD300agene-deficient mice. Statistical significance was determined byStudent's t-test (**: p<0.01, ***: p<0.001).

As shown in FIG. 15, the weight loss due to enteritis induced bydrinking of 2.5% DSS was significantly milder in the CD300agene-deficient mice having no MAIR-I (□) than in the WT mice ().

Example 6B Measurement of Length of Large Intestine (FIG. 16A)

The large intestine was collected from 7 WT mice and 7 CD300agene-deficient mice on Day 6 of the administration of DSS, and thelength from the anal region to the cecum region was measured.Statistical significance was determined by Student's t-test (**:p<0.01).

As shown in FIG. 16A, the CD300a gene-deficient mice had longerlarge-intestines, and large-intestinal atrophy due to the DSS-inducedenteritis was milder in the CD300a gene-deficient mice than in the WTmice.

Example 6C Measurement of Length of Large Intestine (FIG. 16B)

Histological evaluation of the large intestine was simultaneouslycarried out. As a result, it was observed that the CD300a gene-deficientmice had milder histological changes in the large intestine than the WTmice (FIG. 16B).

Example 6D Measurement of Cytotoxic Activity (FIG. 17)

Large-intestine tissues of the WT mice and the CD300a gene-deficientmice on Day 6 of the DSS administration were stained by HE(hematoxylin-eosin) staining, and scored from 0 to 5 (0: no change; 1:hypertrophy and structural changes in crypts; 2: remarkable decrease ingoblet cells; 3: crypts are found, but their structures are hardlymaintained; 4: disappearance of crypts is found, but no detachment ofthe epithelium is found; 5: disappearance of crypts, detachment of theepithelium, and remarkable cellular infiltration are found).

Each of the large intestines of 7 WT mice and 7 CD300a gene-deficientmice was photographed with visual field enhancement at 10 areas from theanal side, and the obtained images were scored. Statistical significancewas determined by Student's t-test (***: p<0.001). The results are shownin FIG. 17.

As shown in FIG. 17, tissue injury due to the DSS enteritis was mild inthe CD300a gene-deficient mice.

Example 6E Measurement of IL-10 (FIG. 18A)

DSS or water (control group) was administered, and the large intestineof each mouse was removed on Day 9 after the administration of DSS. Thelarge intestine was washed with phosphate buffer, and cut into 3-mm³pieces from the anal side.

The minced tissue of the large intestine was cultured in 10% FBS RPMI1640 (GIBCO) for 12 hours, and the supernatant was then collected. Thecollected culture supernatant was subjected to measurement of theprotein contents of cytokines (TNF-α, IL-6 and IL-10) by sandwich ELISA.Statistical significance was determined by Student's t-test (*: p<0.05).

As shown in FIG. 18, in the large-intestine tissue with DSS-inducedenteritis, the contents of IL-6 and TNF-α were not different between theWT mice and the CD300a gene-deficient mice. However, higher-levelproduction of IL-10 was found in the CD300a gene-deficient mice (FIG.18A).

It is known that, in Mreg cells, IL-10 suppresses expression of CD80 andCD86, which are proteins required for activation of inflammatory T cells(Document 33 listed below).

As shown in FIG. 18A, the CD300a gene-deficient mice showed an increasedproduction of IL-10 at the 5% significance level. Thus, it is thoughtthat IL-10 suppresses growth induction of inflammatory T cells andactivation of inflammatory T cells, thereby contributing maintenance ofhomeostasis of the gut immune system.

Example 6F Measurement of Number of Cells of Each Type (FIG. 18B)

In mice on Day 0, Day 2, Day 4 and Day 6 after administration of DSS,changes in the cell populations of CD4⁺ T cells, CD8⁺ T cells, B cells,macrophages and dendritic cells were investigated.

As a result, as shown in FIG. 18B, no difference was found in the numberof cells of each cell population that have moved to the large intestinebetween the WT mice and the CD300a gene-deficient mice.

Example 6G Expression Analysis of CD300a (FIG. 19)

Cells isolated from the large intestine were stained withAPC-Cy7-labeled anti-CD11b (M1/70), PE-Cy7-labeled anti-CD11c (HL3) andBiotin-anti-CD300a (TX41). The cells in the CD11b⁻CD11c⁻ fraction,CD11b⁺CD11c⁻ fraction, CD11b⁻CD11c⁺ fraction, and CD11b⁺CD11c⁺ fractionwere subjected to measurement of expression of CD300a by flow cytometry.The results are shown in FIG. 19.

As shown in FIG. 19, in the large intestinal lamina propria, expressionof CD300a was found in CD11b⁺CD11c⁺ cells, which can be said to bespecial cells.

Example 6H Quantification of Expression in CD11b⁺CD11c⁺ Cells (FIG. 20)

The CD11b⁺CD11c⁺ cells isolated from the large intestine of each mousewere sorted by FACSAria, and mRNA was then extracted from the cells.cDNA was prepared from the extracted mRNA, and the mRNA levels of IL-10,TNF-α and IL-6 were measured by quantitative PCR.

As a result, as shown in FIG. 20, no difference in the mRNA levels ofIL-10, TNF-α and IL-6 could be found in the CD11b⁺CD11c⁺ cells betweenthe WT mice and the CD300a gene-deficient mice.

Example 6I Quantification of Expression in CD4⁺ T Cells (FIG. 21)

CD4⁺ cells isolated from the large intestine of each mouse afteradministration of DSS were sorted by FACSAria, and mRNA was thenextracted from the cells. cDNA was prepared from the extracted mRNA, andthe mRNA levels of IL-10, Foxp3, TGF-β, T-bet, GATA-3 and RORyt weremeasured by quantitative PCR using ABI 7500 fast. Statisticalsignificance was determined by Student's t-test (*: p<0.05, **: p<0.01).

As shown in FIG. 21, the CD300a gene-deficient mice showed higher mRNAlevels of IL-10, Foxp3 and Tgfβ in the CD4⁺ T cells.

(Discussion)

As shown by the results of Examples 6A to 6I, in mice with DSS-inducedenteritis, deficiency of MAIR-I causes production of anti-inflammatorycytokines such as IL-10 (FIG. 21), leading to amelioration of thedisease state of enteritis.

It is thought that the above phenomenon is not due to an increase in acertain cell population (FIG. 18B), but due to regulation of IL-10production via CD300a (MAIR-I) by the CD11b-positive dendritic cells perse or certain cells influenced by those dendritic cells.

That is, it can be predicted that suppression or inhibition of signaltransduction of CD300a (MAIR-I) via CD11b-positive dendritic cells maylead to amelioration of the disease state of enteritis.

<Atopic Dermatitis>

Involvement of CD300a in atopic dermatitis was investigated. Thematerials and methods, and Examples are shown below.

(Experimental Animals)

Balb/c mice were purchased from Clea Japan, Inc., and kept in theinventors' laboratory under approved breeding room conditions. The miceused in this study were 8 CD300a (MAIR-I) gene-deficient mice and 8Balb/c wild-type (WT: Wild Type) mice. During the experimental period,each mouse was provided with food and water ad libitum, and kept undernormal laboratory conditions.

(Percutaneous Sensitization)

Each mouse was mildly anesthetized with isoflurane (Mylan, Osaka,Japan), and the hair on the back was shaved with an electric shaver. Anarea (1 cm²) on the back skin of each mouse was subjected to at least 10times of tape stripping using adhesive cellophane tape.

On the gauze of Band Aid (registered trademark) tape, 100 μg ofovalbumin (OVA) in 100 μL of phosphate buffered saline was placed, andthe resulting tape was applied to the bodies of 5 mice in each groupsubjected to the tape stripping. To the remaining 3 mice, PBS wasapplied with tape. The tape was once replaced on Day 2, and OVAsensitization with the tape was carried out every day for 1 week.

Each mouse was kept without OVA sensitization during Week 2. During Week3, the mouse was subjected again to OVA sensitization in the same manneras described above. At the end of Week 3, each mouse was sacrificed, andsamples for histology and ELISA were collected.

(Number of Times of Scratching Behavior)

The number of times of scratching behavior was counted by carefulobservation of each mouse for 30 minutes at the end of each of Week 1 toWeek 3.

(Histology)

For histological observation, the skin of the OVA-sensitized area ineach mouse was collected. Each collected skin sample was cut into smalltissue blocks, and immersed in 4% paraformaldehyde at 4° C. for 24hours.

After this fixation, a dehydration step was carried out. All skinsamples were then quickly frozen in acetone in a container containingdry ice. The samples were stored at −30° C. until use.

The skin samples were then cut into sections with a thickness of 4 μmusing a frozen section preparation apparatus, Coldtome HM560E(manufactured by Carl Zeiss, Jena, Germany), and placed on slide glassesprecoated with New Silane III (Muto Pure Chemicals, Co., Ltd., Tokyo,Japan).

The tissue sections were stained with hematoxylin-eosin (manufactured bySakura Finetek Japan), and then stained with toluidine blue(manufactured by Santa Cruz Biotechnology, Inc.).

Initial evaluation of the tissue was performed for the skin depth (celllayer); infiltration of eosinophils, monocytes and mast cells; and thelevel of hyperplasia of fibroblasts.

Example 7A Number of Times of Scratching Behavior (FIG. 22)

FIG. 22 shows the number of times of scratching behavior per 30 minutesin each mouse. Pruritus (a disease that causes itch without causingeruption) is a common condition for atopic dermatitis. Therefore, theevaluation was carried out by counting the number of times of scratchingbehavior by careful observation of each animal for 30 minutes during theOVA sensitization.

As shown in FIG. 22, the OVA-sensitized WT mice (♦) showed a severecondition of scratching behavior after the OVA sensitization, and alarger number of times of scratching behavior than the OVA-sensitizedCD300a gene-deficient mice (▴). The largest number of times ofscratching behavior was observed at the end of the 4th OVAsensitization.

During the OVA sensitization, the most severe symptom of scratchingbehavior was observed in the WT mice (FIG. 22). Appearance of a severesymptom of scratching behavior is one of the most common pathologicalfeatures of atopic dermatitis. Thus, involvement of CD300a(MAIR-I)-positive cells in an important role in atopic dermatitis mightbecome clear.

Example 7B Observation (See FIG. 23)

The skin of each OVA-sensitized mouse was observed.

FIG. 23(A) shows the skin of a WT mouse that had not undergone OVAsensitization. In untreated WT mice, which had not been subjected toovalbumin sensitization, cellular infiltration was not found in thedermis, and a thin epidermis could be observed. The lower panels in FIG.23 show magnified views of the rectangular areas in the upper panels.The length of each thick bar corresponds to 10 μm.

On the other hand, as shown in FIG. 23(B), the skin of theOVA-sensitized WT mice showed hyperplasia of the epidermis andhyperplasia of fibroblasts. Obvious infiltration of eosinophils (seewhite arrowheads) and monocytes was found in the dermis of the skin inthe WT mice subjected to sensitization with ovalbumin.

As shown in FIG. 23(C), in the epidermis of the CD300a gene-deficientmice that had not been subjected to OVA sensitization, cellularinfiltration was not found, and a thin epidermis could be observedsimilarly to FIG. 23(A).

Surprisingly, as shown in FIG. 23(D), the skin of the OVA-sensitizedCD300a gene-deficient mice showed an increased thickness of theepidermis, but did not show hyperplasia of the epidermis. Further, thedermis showed neither cellular infiltration nor hyperplasia offibroblasts.

Example 7C Staining of Mast Cells (See FIG. 24)

As shown in FIG. 24, all mouse skin samples were subjected to toluidineblue staining. Toluidine blue staining is a staining method thatpreferentially stains intracellular granules in mast cells.

FIG. 24(A) shows the skin of a WT mouse that had not been subjected toOVA sensitization. FIG. 24(B) shows the skin of an OVA-sensitized WTmouse. FIG. 24(C) shows the skin of a CD300a gene-deficient mouse thathad not been subjected to OVA sensitization. FIG. 24(D) shows the skinof an OVA-sensitized WT mouse. Stained mast cells are indicated by openarrowheads.

As shown in FIG. 24, after the OVA sensitization, an increased number ofmast cells were found in the skin of the WT mouse. Moreover, theepidermis in the mouse was thicker than that in the CD300agene-deficient mouse.

Example 7D Measurement of Number of Cell Layers (FIG. 25A)

All mouse skin samples were subjected to counting of the number of celllayers in the epidermis (see FIG. 25A). After the OVA sensitization, theWT mice showed the largest number of cell layers. On the other hand, inthe OVA-sensitized CD300a gene-deficient mice, the number of cell layerswas less than half of this number.

Example 7E Counting of Number of Eosinophils and Number of Mast Cells(FIG. 25B)

As shown in FIG. 25B, all mouse epidermal samples were subjected tocounting of the number of eosinophils and the number of mast cellsshowing infiltration into the dermis, which are indices for atopicdermatitis. The largest cell numbers were observed in the epidermis ofthe WT mice after the sensitization with ovalbumin.

On the other hand, in the OVA-sensitized CD300a gene-deficient mice, theincreases in the number of eosinophils and the number of mast cells weremilder than those in the WT mice. After the OVA sensitization,hyperplasia of epidermis and hyperplasia of fibroblasts appeared mostseverely in the WT mice (FIG. 23 and FIG. 25A). Hyperplasia of epidermisand fibroblasts is another major pathological feature of atopicdermatitis.

Further, a high level of infiltration of eosinophils, mast cells andmonocytes was found in the skin of the OVA-sensitized WT mice (FIG. 23to FIG. 25B). Interaction between infiltrating eosinophils andhyperplasia of fibroblasts causes secretion of IL-31. IL-31 is anitch-inducing cytokine (Document 34 listed below: Wong C K et al.,2012).

Therefore, after OVA sensitization, WT mice show more severe features ofatopic dermatitis than CD300a gene-deficient mice.

(Immunohistology)

In order to detect CD300a (MAIR-I) and the Langerin antigen on serialsections of the skin, the single-step or two-step method of enzymeimmunohistochemistry was carried out. First, all sections were rinsed 3times with phosphate-buffered saline supplemented with 0.05% Tween(TPBS, pH 7.4). The sections were then immersed for 30 minutes in eachof cold absolute methanol and 0.5% H₂O₂.

After the washing in TPBS, the sections were subjected to blockingtreatment using the Blocking One Histo reagent (Nacalai Tesque, Inc.,Kyoto, Japan) for 10 minutes, and then washed with TPBS. The skinsections were cultured at 4° C. for 18 hours in the presence ofanti-Langerin goat IgG (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif., US) and biotinylated anti-CD300a (MAIR-I) rat IgG 2aλ (preparedin the inventors' laboratory), and then at room temperature for 1 hourin the presence of biotinylated donkey anti-goat IgG as the secondaryantibody for Langerin.

Finally, these were cultured in the presence of DAB/Metal Concentrate(Thermo Scientific, Waltham, Mass., US), and counterstained withhematoxylin. Negative control sections were cultured in the presence ofTPBS or an isotype control antibody instead of the primary antiserum.

Example 7F Langerin Immunostaining (FIG. 26)

The epidermis of each mouse was investigated to see whether or not it ispositive for Langerin, which is a dendritic cell marker inimmunostaining. Langerin is a C-type lectin expressed in Langerhanscells, and also expressed in part of dermal dendritic cells. Langerin isinvolved in recognition and incorporation of antigens, and formation ofBirbeck granules, which are responsible for intracellular antigendelivery.

(Results)

FIG. 26(A) shows the epidermis of an untreated WT mouse (mouse withoutOVA sensitization). FIG. 26(B) shows the epidermis of an OVA-sensitizedWT mouse. FIG. 26(C) shows the epidermis of an untreated CD300agene-deficient mouse. FIG. 26(D) shows the epidermis of anOVA-sensitized CD300a gene-deficient mouse. The panels a, b, c and d aremagnified views of the rectangular areas in the upper panels.

After the OVA sensitization, a significantly larger number ofLangerin-positive cells were found in the epidermis of WT mice than inthe epidermis of CD300a gene-deficient mice. The arrowheads indicateLangerin-positive cells. Each scale bar represents 10 μm.

Example 7G Counterstaining after Langerin Immunostaining (FIG. 27)

Immunopositivity of the Langerin antibody was evaluated bycounterstaining with toluidine blue. The skin of the WT mouse showed anincreased number of Langerin-positive cells in the dermis. In thedermis, interaction of several Langerin-positive cells with mast cellswas found. Mast cells were stained in purple. Each scale bar represents10 μm.

Langerhans cells and skin dendritic cells are major antigen-presentingcells in the skin. Langerhans cells are positive for the Langerinantigen in their cell membranes, and Langerin-positive cells are alsopresent among skin dendritic cells (Document 35 listed below: NakajimaS. et al, 2012).

Skin Langerin-positive dendritic cells interact with mast cells toactivate CD4-positive T cells (Document 36 below: Otsuka A. et al.,2011). In the OVA-sensitized model, the numbers of mast cells (FIG. 24and FIG. 25B) and Langerin-positive cells (see FIG. 26) largelyincreased in the skin of the OVA-sensitized NT mice (see whitearrowheads in FIG. 26 for comparison).

In the skin of the OVA-sensitized NT mice, interaction between mastcells and Langerin-positive cells was found (FIG. 27).

(Discussion)

Thus, it can be deduced that atopic dermatitis more severely appears inthe skin of WT mice than the skin of CD300a gene-deficient mice. Theseresults suggest that CD300a (MAIR-I) plays an important role in atopicdermatitis.

In the dermis of the WT skin, CD300a (MAIR-I)-positive cells largelyincreased after the OVA sensitization (FIG. 27). This result furtherconfirms that CD300a (MAIR-I)-positive cells play an important role inatopic dermatitis.

(Treatment of Atopic Dermatitis)

(Treatment with Anti-CD300a (MAIR-I) Antibody)

In the present experiment, 6 Balb/c mice of 7 weeks old were used.According to the protocol shown in FIG. 29, 3 animals out of the 6animals were subjected to intravenous injection of anti-CD300a (MAIR-I)rat IgG 2aλ (TX41), and the remaining 3 animals were subjected tointravenous injection of a rat IgG 2aλ control antibody (TX74). Both ofthese antibodies were prepared and checked in the inventors' laboratory.“TX74” is an isotype control antibody of TX41, and does not have aneutralizing action as described below. Each antibody was diluted withsterile PBS to an antibody concentration of 1600 μg/mL, and 150 μL ofthe dilution was injected at once.

<Blocking of CD300a (MAIR-I) Antigen by Injection of CD300a (MAIR-I)Antibody>

FIG. 29 shows the procedure to block the CD300a (MAIR-I) antigen in aBalb/c WT mouse. Each thick line indicates the period of OVAsensitization. The arrowheads indicate the schedule of injection of theantibodies.

Total serum IgE was evaluated at the end of each week of sensitization.Histological samples were collected after the continuous sensitization.The anti-rat CD300a (MAIR-I) antibody IgG 2aλ (TX41), and the controlantibody (TX74), which is an isotype thereof, were used for intravenousinjection.

(ELISA)

Peripheral blood was collected from the retro-orbital cavity using aplain glass hematocrit tube (Drummond Scientific Company, Broomall, Pa.,US), and centrifuged at 12000 rpm for 5 minutes.

Serum was collected by cutting the tube, and the whole serum was dilutedwith a blocking serum before use in ELISA. The ELISA experiment wascarried out according to the standard protocol for total IgE,recommended by BD Biosciences, California, US.

Example 7H Immunoreaction with Anti-CD300a Antibody (Confirmation ofPresence of Receptors) (FIG. 28)

Immunopositivity of an anti-CD300a antibody was evaluated with a skinsample of each mouse. FIG. 28(A) shows untreated epidermis of a WTmouse, and (B) shows OVA-sensitized epidermis of a WT mouse.

The epidermis of WT mice after OVA sensitization showed cells that aresignificantly immunopositive. The epidermis of CD300a gene-deficientmice did not show immunopositive reaction. The arrowheads in (B)indicate CD300a (MAIR-I)-positive cells. Each scale bar represents 10μm.

Example 7I Treatment by Administration of CD300a Antibody (FIG. 30)(ELISA)

As shown in FIG. 29, each of TX41 and TX74 was administered to WT miceaccording to the above-described procedure, and the IgE level, which isan index of atopic dermatitis, was measured.

FIG. 30 shows the total serum IgE level measured by ELISA. The IgE levelafter OVA sensitization was higher in the mice to which TX74 wasinjected than in the mice to which TX41 was injected.

Example 7J Treatment by Administration of Anti-CD300a Antibody (FIG. 31)

As shown in FIG. 31, after the OVA sensitization, the WT mice to whichTX74 was injected showed a more severe scratching behavior than the WTmice to which TX41 was injected.

Example 7K H&E Staining (FIG. 32)

As shown in FIG. 32, skin sections of the WT mice in Example 7I weresubjected to H&E staining. After the OVA sensitization, the skin of theWT mice to which TX74 was injected showed higher levels of hyperplasiaof the epidermis and infiltration of monocytes than the skin of the WTmice to which TX41 was injected. In FIG. 32, the scale bar in the lowerright corner of each photograph represents 10 μm.

Example 7L Toluidine Blue Staining (FIG. 33)

As shown in FIG. 33, the skin of each WT mouse was subjected totoluidine blue staining. After the OVA sensitization, the skin of the WTmice to which TX71 was injected showed more mast cells than the skin ofthe WT mice to which TX41 was injected. Each scale bar represents 10 μm,similarly to FIG. 32.

(Discussion)

The total IgE and the number of scratching behavior were higher in themice to which TX74 was injected than the mice to which TX41 was injected(see FIG. 30 and FIG. 31). The epidermal thickness, number ofinfiltrating cells, number of fibroblasts and number of mast cells werealso higher in the mice to which TX74 was injected than the mice towhich TX41 was injected (see FIG. 32 and FIG. 33).

From these results, it could be further confirmed that CD300a (MAIR-I)plays an important role in atopic dermatitis, and the effect of TX41,which is an anti-CD300a antibody, as a therapeutic agent for atopicdermatitis could be confirmed.

<Celiac Disease>

Celiac disease (CD) is a progressive enteritis caused by immune responseto dietary gluten protein. The adaptive immune response specific to thegliadin peptide derived from gluten is involved in the progression ofceliac disease. The innate immune response as the fundamental cause ofthe disease has not been completely elucidated.

Here, we demonstrate that CD300a (MAIR-I), which is expressed in laminapropria macrophages (macrophages in the lamina propria) and is a memberof the bone marrow-associated immunoglobulin-like receptor family, playsa regulatory role in the progression of dietary gluten-inducedintestinal diseases.

CD300a gene-deficient mice, which lack CD300a (MAIR-I), fed with ahigh-gluten diet showed celiac disease-like symptoms. Compared towild-type (WT) mice fed with a high-gluten diet, each CD300agene-deficient mouse showed a mild increase in the body weight, highclinical score, large amount of transglutaminase 2, and accumulation oflamina propria macrophages in the jejunum.

Compared to the WT mice fed with a high-gluten diet, lamina propriamacrophages of each CD300a gene-deficient mouse fed with a high-glutendiet showed higher expression of IL-6, IL-15, TNF-α, IFN-β, MCP1 andMCP5.

After in vitro stimulation with the toxic gliadin peptide (P31-43),lamina propria macrophages of each CD300a gene-deficient mouse showedhigher-level expression of the above-described cytokines.

Enhanced expression of IL-6, TNF-α and IFN-β occurred in lamina propriamacrophages of CD300a gene-deficient mice, which lack CD300a (MAIR-I)and have a MyD88-deficient and TRIF-deficient genetic background.

Further, blocking of binding of phosphatidyl serine, which is the ligandof CD300a (MAIR-I) on apoptotic cells, to CD300a (MAIR-I) promotedproduction of cytokines in lamina propria macrophages of WT mice.

In summary, these results indicate that the interaction betweenmacrophages in the intestinal lamina propria and CD300a (MAIR-I) onapoptotic cells plays a protective role in progression of celiacdisease, by MyD88- and TRIF-mediated inhibitory gliadin signalingpathways.

(Materials and Methods) (Mice)

Male Balb/c wild-type (WT) mice of 9 to 14 weeks old, and littermates ofBalb/c CD300a gene-deficient mice, which do not have CD300a (MAIR-I),were provided. These mice were used in an experiment in which they werefed with a normal diet (ND), high-gluten diet (HGD) or gluten-free diet(GFD).

For in vitro experiments, Balb/c WT mice; and CD300a gene-deficientBalb/c or C57BL/6 B6 mice, which have no CD300a (MAIR-I); were used.These mice had the same sex and age. MyD88-gene deficient B6 mice andTRIF gene-deficient B6 mice were purchased from Oriental Bioscience(Kyoto, Japan). All mice were kept under specific pathogen-freeconditions.

(Feeding Test)

WT mice and CD300a (MAIR-I) mice were kept with normal powder dietcontaining less than 2% gluten (MF, Oriental Yeast Co., Ltd., Tokyo,Japan) (ND). WT mice and CD300a gene-deficient mice were kept with ahigh-gluten diet (HGD), which is the same as MF except that gluten iscontained at 30% (Sigma-Aldrich, St. Louis, Mo.). In severalexperiments, mice were fed with pellets of ND (MF) or a gluten-free diet(GFD; AIN-76A, Research Diets, Inc., New Brunswick, N.J.).

(Depletion of Microbiota)

Depletion of the microbiota was carried out as in the [Document 37listed below]. Depletion of the microbiota was confirmed by observingcolony forming units in feces from each mouse using an agarose platecontaining brain-heart infusion medium.

(Histopathological Analysis)

Histopathological changes in mice were observed in the later-describedspecific weeks after feeding with ND, HGD or GFD. Samples of the jejunumand colon were isolated and fixed with formalin, followed by stainingwith hematoxylin-eosin.

The standard for the clinical score for the jejunum was defined as inthe [Document 38 listed below]. Briefly, the score of the ratio betweencrypts and villi was calculated according to the average depths ofcrypts and villi (score 0 to 3). Further, the score (score 0 to 3) ofinfiltration of monocytes was calculated according to the averagediameters of the lamina propria of villi, and crypts. The final clinicalscore was represented as the total of 0 to 6.

The number of intraepithelial lymphocytes in intestinal epithelial cells(IELs) of the jejunum was counted, and represented as the IEL number per100 intestinal epithelial cells ([Document 39 listed below]).Quantification of transglutaminase 2 (TG2) in the jejunum was carriedout using a tissue suspension of the jejunum. The test was carried outusing TG2-CovTest (Zedira, Darmstadt, Germany) according to themanufacturer's instructions.

(Titration of IgG and IgA Antibodies Against Gliadin)

The serum antibody titers of IgG and IgA against gliadin in mice weredetermined by an enzyme immunoassay. This was carried out by the methoddescribed in the [Document 40 listed below] with some modifications.

An anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) (GEHealthcare, Little Chalfont, UK), and an anti-mouse IgA antibody labeledwith HRP (Southern Biotech, Birmingham, Ala.) were used as anti-gliadinIgG and IgA detection antibodies, respectively. OPD Reagent(Sigma-Aldrich) was used as the substrate of HRP in colorimetricanalysis.

(Isolation of Lamina Propria Macrophages and Dendritic Cells)

Lamina propria macrophages (LP Mφ) and dendritic cells (DCs) wereisolated according to the [Document 41 listed below] with somemodifications.

After removal of the mesentery and Peyer patches, the jejunum was cutinto small pieces. The pieces were washed a total of 3 times with PBSbuffer supplemented with 2 mM EDTA (Sigma Aldrich) and 20% FCS, withshaking at 37° C. for 15 minutes.

The remaining tissue was homogenized, and then digested in PBS buffersupplemented with 1.5 mg/mL type VIII collagenase (Sigma-Aldrich) and20% FCS, with shaking at 37° C. for 20 minutes.

Lamina propria macrophages and lamina propria dendritic cells expressingCD45 in the tissue suspension were concentrated using biotinylatedanti-mouse CD45 (30-F11, BD Biosciences, Franklin Lakes, N.J.) andstreptavidin particles plus IMAG (BD Biosciences).

The CD45-expressing cells in the lamina propria were stained with afluorescein isothiocyanate-conjugated anti-mouse CD11b (M1/70)phycoerythrin-conjugated CD11c (HL3) propidium iodide, Alexa 647-labeledTX41 (anti-mouse CD300a (anti-mouse MAIR-I) rat IgG2a) or Alexa647-labeled TX74 (anti-FLAG, isotype control for TX41) biotinylatedanti-mouseCD45, and streptavidin allophycoerythrin Cy7(allophycoerythrin-Cy7).

All fluorescently labeled antibodies and streptavidinallophycoerythrin-Cy7 were purchased from BD Biosciences.

CD11b⁺CD11c^(low) cells and CD11c⁺ cells gated based on the number ofCD45⁺PI⁻ cells were isolated as lamina propria macrophages and laminapropria dendritic cells using FACSAria (BD Biosciences).

An important principle of flow cytometry data analysis is to selectivelyvisualize the particles of interest while removing unnecessary particles(dead cells, residues and the like). This operation is called gating(gate).

(Flow Cytometry Analysis)

Macrophages, CD11b⁺ dendritic cells and CD11b⁻ dendritic cells among thecells of lamina propria, which are CD45⁺PI⁻, were gated based on thenumbers of CD11b⁺CD11c^(low), CD11b⁺CD11c⁺ and CD11b⁻CD11c⁺,respectively.

Intraepithelial lymphocytes and intestinal epithelial cells wereobtained by washing small pieces of jejunum with PBS buffer supplementedwith 2 mM EDTA and 20% FCS. The intraepithelial lymphocytes andintestinal epithelial cells contained in the suspension were gated basedon the number of CD45′PI cells and the number of CD45 PI cells.

Expression of CD300a (MAIR-I) in macrophages, CD11b⁺ dendritic cells andCD11b⁻ dendritic cells in the lamina propria, intraepitheliallymphocytes, and intestinal epithelial cells were analyzed with Alexa647-labeled TX41 (anti-mouse MAIR-I, rat IgG2a) or Alexa 647-labeledTX74 (anti-FLAG of TX41, isotype control).

Apoptotic cells presenting phosphatidyl serine (PS) were analyzed usingallophycocyanin-conjugated annexin V. Flow cytometry analysis wascarried out using FACSAria (BD Biosciences).

(Quantitative RT-PCT Method)

In Isogen LS (Nippon Gene, Tokyo, Japan), 10000 to 20000 lamina propriamacrophages and lamina propria dendritic cells that were stimulated withor not stimulated with the toxic gliadin peptide P31-43 wereresuspended, and total RNA was isolated from the resulting suspensionaccording to the manufacturer's instructions.

Single-stranded DNA was synthesized from total RNA using a cDNA reversetranscription kit (Applied Biosystems, Foster City, Calif.). The primersets used for quantitative reverse transcriptase-mediated polymerasechain reaction (Q-RT-PCR) were designed by PrimerBank(http://pga.mgh.harvard.edu/primerbank/) [Document 42 listed below].

Q-RT-PCR was carried out using Platinum SYBR Green Super Mix UDG(Invitrogen, Carlsbad, Calif.) and ABI 7500 Fast (Applied Biosystems).The data were analyzed by the DDCT (delta delta CT) method.

Control samples expressing all genes tested in this study were preparedfrom spleen cells stimulated with lipopolysaccharide (1000 ng/mL, 6hours), and were single-stranded DNA. The results were shown based oncomparative quantification with samples showing expression of therespective genes.

(Gliadin Stimulation)

The toxic α-gliadin peptide P31-43 derived from gluten [Document 43listed below] and the ovalbumin peptide P323-339 as a control weresynthesized by Operon Biotechnologies (Huntsville, Ala.).

The purities of these peptides were not less than 95%, and the endotoxinunit/mL was less than 0.001. This was confirmed with Limulus Color KYTest Wako (Wako Pure Chemical Industries, Ltd., Osaka).

Macrophages in thioglycolate-induced peritoneal exudate cells (PECs)prepared from a B6 mouse were stimulated using 100 μg/mL gliadin peptideP31-43 or ovalbumin P323-339 as described in the [Document 44 listedbelow].

In wells of a 96-well flat-bottom plate, 10000 to 20000 lamina propriamacrophages and CD11b⁺ dendritic cells derived from WT mice or CD300agene-deficient mice were cultured in RPMI 1640 medium supplemented with10% FCS, glutamine/streptomycin/penicillin, HEPES and non-essentialamino acids.

Lamina propria macrophages of Balb/c mice or B6 mice were stimulated for10 hours or 3 hours with 100 μg/mL gliadin peptide P31-43. In severalexperiments, the lamina propria macrophages were treated or not treatedwith MFG-E8 (final concentration, 5 μg/mL) of a recombinant mouse at 4°C. for 30 minutes, and further, stimulated with the gliadin peptideP31-43 at 37° C. for 3 hours.

(Statistical Analyses)

All statistical analyses were carried out using the Mann-Whitney U test.Statistical significance was judged at P<0.05.

Example 8A Body Weight (BW) Change (FIG. 34)

In order to investigate whether or not CD300a (MAIR-I) is involved inthe progression of celiac disease (CD), CD300a gene-deficient Balb/cmice or wild-type (WT) mice were kept with a normal diet containing lessthan 2% gluten (ND) or a high-gluten diet containing 30% gluten (HGD).After starting feeding with the normal diet or high-gluten diet, changesin the body weight (BW) were monitored.

“∘” represents “WT mice, n=7”; “” represents “CD300a gene-deficientmice, normal diet, n=9”; “□” represents “WT mice, high-gluten diet,n=10”; and “▪” represents “CD300a gene-deficient mice, high-gluten diet,n=14”.

The CD300a gene-deficient mice kept with the high-gluten diet (HGD)showed a significantly smaller degree of increase in the body weight(BW) than the WT mice kept with the high-gluten diet (HGD) (FIG. 34).The CD300a gene-deficient mice were confirmed to show progression ofceliac disease-like enteropathy after feeding with the high-gluten diet.These results suggest that CD300a (MAIR-1) plays a protective role inthe progression of the intestinal disease in the jejunum after feedingwith a high-gluten diet.

Example 8B HSE Staining (FIG. 35)

Histopathological analysis of the intestines of mice at Week 20 afterfeeding with a normal diet or a high-gluten diet (HGD) was carried out.The jejunum of each mouse was fixed with formalin, followed by stainingwith hematoxylin-eosin. FIG. 35 shows a representative result obtainedfor a mouse in each group.

The CD300a gene-deficient mice kept with a high-gluten diet (HGD) showeda more advanced intestinal disease in the jejunum compared to the WTmice kept with a high-gluten diet (HGD) (see FIG. 35)

Example 8C Number of Intraepithelial Lymphocytes (FIG. 36, FIG. 37)

According to the above-described method, the clinical score and thenumber of intraepithelial lymphocytes (IELs) per 100 intestinalepithelial cells (IECs) in the jejunum of each mouse were counted atWeek 20 after feeding with the normal diet or the high-gluten diet. “*and **” represent p<0.05 and p<0.01, respectively.

As described above, the CD300a gene-deficient mice showed atrophy ofvilli in the jejunum (FIG. 35), and significantly higher clinical scores(see FIG. 36). Further, an increase in intraepithelial lymphocytes(IELs) in small-intestinal epithelial cells (IECs) was found as comparedto the CD300a gene-deficient mice kept with FD as well as the WT micekept with a high-gluten diet (HGD) (see FIG. 37).

Example 8D TG2-CovTest (Colorimetric Analysis for Quantitation ofSpecific TG2 Cross-Linking Activity) (FIG. 38)

The level of transglutaminase 2 (TG2) is known to be influenced byintestinal inflammation. TG2 is a key enzyme for the progression ofceliac disease. WT mice and CD300a gene-deficient mice were fed with anormal diet or high-gluten diet for at least 20 weeks, and the level oftransglutaminase 2 (TG2) in a jejunum suspension of each mouse wasquantified with TG2-CovTest.

As a result, the TG2 level in the intestine was highest in the CD300agene-deficient mice kept with a high-gluten diet (HGD) among the groupsof mice (see FIG. 38).

Example 8E Flow Cytometry (FIG. 39)

Expression of CD11b and CD11c in lamina propria cells gated for theCD45⁺PI⁻ cell population was analyzed by flow cytometry. Lamina propria(LP) cells of WT mice or CD300a gene-deficient mice of the Balb/c strainfed with the normal diet or high-gluten diet were analyzed by flowcytometry. CD45-expressing LP cells were concentrated using the I-Nagtechnology (cell separation system using magnetic beads).

CD11b⁺CD11c^(low) cells are lamina propria macrophages, and CD11b⁺CD11c⁺and CD11b⁻CD11c⁺ are lamina propria (LP) dendritic cells (DCs).

Example 8F Quantitative and Qualitative Changes in Immunocytes (FIG. 40)

In each of WT mice and CD300a gene-deficient mice, the frequency oflamina propria (LP) macrophages (open bar), the frequency of laminapropria (LP) CD11b⁺ dendritic cells (closed bar), and the frequency oflamina propria (LP) CD11b⁻ dendritic cells (shaded bar) were measured.

That is, quantitative and qualitative changes in the immunocytes in thelamina propria of the jejunum of NT mice and CD300a gene-deficient micekept with a normal diet (ND) or high-gluten diet (HGD) wereinvestigated.

The frequency of lamina propria macrophages expressing CD11b⁺CD11c^(low)(lp Mf) was significantly higher in the CD300a gene-deficient mice keptwith a high-gluten diet (HGD) than in the WT mice kept with ahigh-gluten diet (HGD) or in the CD300a gene-deficient mice kept with anormal diet (ND) (see and compare the cell populations 5.8 in the lowerright panel, 2.3 in the lower left panel, and 2.2 in the upper rightpanel of FIG. 39; and see FIG. 40).

Lamina propria macrophages in the intestine are cells differentiatedfrom inflammatory monocytes expressing CCR2 in peripheral blood[Document 45 listed below].

In view of the fact that CCR2 is a receptor of MCP1 and MCP5, theincreased expression of these chemokines in lamina propria macrophagesafter stimulation with gliadin is consistent with the high frequency oflamina propria macrophages in the CD300a gene-deficient mice kept with ahigh-gluten diet (HGD) (see FIG. 40).

Example 8H Gene Expression Levels of Cytokines and Chemokines (FIG. 41)

The gene expression levels of cytokines and chemokines in lamina propria(LP) macrophages isolated from each mouse were analyzed by quantitativeRT-PCR. In FIG. 41, the expression level of each gene is expressed asthe relative level with respect to the level in WT mice fed with anormal diet, which is taken as “1” to be used as a standard.

Lamina propria macrophages in the CD300a gene-deficient mice kept with ahigh-gluten diet (HGD) showed significantly higher levels ofinflammatory cytokines (IL-6, IL-15, TNF-α, IFN-b and MCP1) and somechemokines such as MCP5 compared to the WT mice kept with a high-glutendiet (HGD) (see FIG. 41).

The enhanced expression of inflammatory cytokines and chemokines fromlamina propria macrophages in the CD300a gene-deficient mice kept with ahigh-gluten diet (HGD) is thought to be due to the intestinal diseaseobserved in the mice.

Further, interestingly, lamina propria macrophages in the CD300agene-deficient mice kept with a normal diet (ND) showed higherexpression of IL-15 and p19 compared to the WT mice kept with ND (FIG.48).

Example 8I FIG. 42

Expression of CD300a (MAIR-I) in lamina propria macrophages, CD11b⁺dendritic cells, CD11b⁻ dendritic cells, intestinal epithelial cells andintraepithelial lymphocytes was analyzed by flow cytometry.

CD300a (MAIR-I) was detected using TX41 (anti-MAIR-I mouse monoclonalantibody). Lamina propria macrophages (M_(T)), CD11b⁺ dendritic cellsand CD11b⁻ dendritic cells were gated based on the number of CD45⁺PI⁻lamina propria cells.

Intraepithelial lymphocytes (IELs) and intestinal epithelial cells(IECs) were gated into the CD3⁺CD45⁺PI⁻ and CD45⁻PI⁻ fractions,respectively.

CD300a (MAIR?I) is expressed in most of bone marrow cells includingmacrophages, granulocytes, mast cells and dendritic cells ([Document 46listed below]).

As described above, the present inventors analyzed expression of CD300a(MAIR-I) in macrophages on the lamina propria, CD11b⁺ inflammatorydendritic cells in the lamina propria, CD11b⁻ tolerogenic dendriticcells, intestinal epithelial cells (IELs) and intestinal epithelialcells (IECs), and lamina propria macrophages, and further investigatedthe CD300a (MAIR-I)-expressing subpopulations of lamina propria CD11b⁺dendritic cells and lamina propria CD11b⁻ dendritic cells. As a result,no expression of CD300a (MAIR-I) was found in the intestinal epithelialcells (IELs) and intestinal epithelial cells (IECs) (FIG. 42).

Example 8J FIG. 43

Although the receptor of the gliadin peptide has not been identified,earlier studies reported that the gliadin P31-43 peptide accumulates inearly endosomes and inhibits their maturation (Documents 47 and 48listed below).

Lamina propria (LP) CD11b⁺ dendritic cells were isolated from Balb/c WTmice or CD300a gene-deficient mice, and stimulated in vitro with 100mg/mL toxic gliadin peptide P31-43 for 10 hours.

The gene expression levels of cytokines and chemokines (IL-6, IL-15,TNF-α, IFN-β, MCP1 and MCP5) in lamina propria (LP) CD11b⁺ dendriticcells in the WT mice and the CD300a gene-deficient mice after thegliadin stimulation were analyzed by quantitative RT-PCR. The expressionlevel of each gene is expressed as the relative level with respect tothe level in untreated WT mice, which is taken as “1” to be used as astandard.

As a result of the investigation of the gene expression levels, theexpression levels of IL-6, IL-15, TNF-α, IFN-β, MCP1 and MCP5 afterstimulation with the gliadin peptide P31-43 were significantly higher inlamina propria macrophages of CD300a gene-deficient mice than in laminapropria macrophages of WT mice (FIG. 43).

Inflammatory lamina propria CD11b⁺ dendritic cells of CD300agene-deficient mice kept with a normal diet (ND) showed higherexpression of IL-15 as compared to WT mice kept with a normal diet (ND).

Example 8K Suppression of Gliadin-Induced Expression of Cytokines (FIG.44)

The present inventors tested whether or not addition of a recombinantmouse MFG-E8 protein can block the interaction between CD300a (MAIR-I)on lamina propria macrophages and phosphatidyl serine (PS) presented onapoptotic cells.

Macrophages in lamina propria cells derived from B6 WT mice or CD300agene-deficient mice were pretreated using or without using 5 mg/mL mouseMFG-E8, and then stimulated in vitro with the toxic gliadin peptideP31-43 for 10 hours.

Expression of the genes for IL-6, TNF-α and IFN-β in the stimulatedmacrophages was then analyzed by quantitative RT-PCR. The expressionlevel of each gene was determined by comparative quantification in whichthe expression level in the untreated WT mice was taken as “1”.

As a result, as compared to the untreated lamina propria macrophages ofWT mice, the lamina propria macrophages of the WT mice treated withMFG-E8 significantly increased expression of IL-6, TNF-α and IFN-β (FIG.44).

These results indicate that the interaction between CD300a (MAIR-I) onlamina propria macrophages and phosphatidyl serine on apoptotic cellssuppresses the MyD88-mediated and TRIF-mediated gliadin signalingpathways to regulate excessive inflammation reaction against nutritiongluten.

There results suggest that the interaction between CD300a (MAIR-I) onlamina propria macrophages and phosphatidyl serine on apoptotic cellssuppresses the MyD88-mediated and TRIF-mediated gliadin signalingpathways to thereby regulate excessive inflammation reaction againstdietary gluten.

Example 8L FIG. 45

Balb/c WT mice and CD300a gene-deficient mice fed with a normal diet ora high-gluten diet were subjected to histopathological analysis of thelarge intestine.

As a result, no atrophic change was observed in the intestine of theCD300a gene-deficient mice kept with a high-gluten diet (HGD) (FIG. 45).

Example 8M FIG. 46

Sera derived from Balb/c WT mice and CD300a gene-deficient mice fed witha normal diet or a high-gluten diet were subjected to investigation ofthe gliadin IgG and IgA antibody titers according to the above-describedmethod.

In FIG. 46, the upper panels show data obtained for the individual mice,and the lower panels show the average values.

As a result, the CD300a gene-deficient mice showed enteritis afteringestion of the high-gluten diet, but no increase in the IgG and IgAtiters against gliadin was found.

Example 8N Pathological Analysis (FIG. 47)

WT mice and CD300a gene-deficient mice fed with a gluten-free diet weresubjected to pathological analysis.

A gluten-free diet was fed to Balb/c WT mice and CD300a gene-deficientmice. From the start of feeding with a gluten-free diet, changes in thebody weight (BW) of each mouse were monitored over time.

As a result, the CD300a gene-deficient mice showed a significantly lowerrate of increase in the body weight than the WT mice.

Example 8O Expression Levels of Cytokines (FIG. 48)

The expression levels of cytokines in CD11b⁺ dendritic cells andmacrophages in the lamina propria (LP) of WT mice and CD300agene-deficient mice under normal conditions were investigated.

Lamina propria (LP) macrophages were isolated from Balb/c WT mice andCD300a gene-deficient mice, and expression of p19 in the macrophages wasanalyzed by quantitative RT-PCR. The gene expression level was expressedas the relative level with respect to the level in the WT mice, whichwas regarded as “1”.

Further, similarly, lamina propria (LP) CD11b⁺ dendritic cells wereisolated from Balb/c mice, and expression of IL-15 in the cells wasanalyzed by quantitative RT-PCR. The expression level was expressed asthe relative level with respect to the level in the WT mice, which wastaken as “1”.

As shown in FIG. 48, the CD300a gene-deficient mice showed significantlyhigher expression levels of the p19 and IL-15 genes than the WT mice.

Example 8P FIG. 49

Expression of cytokines in macrophages in thioglycolate-inducedperitoneal exudate cells after stimulation with the toxic gliadinpeptide P31-43 was investigated.

In view of the fact that only lamina propria macrophages in CD300agene-deficient mice produce a large amount of inflammatory cytokinesafter feeding with a high-gluten diet (HGD), the present inventorsinvestigated whether or not the gluten-derived toxic gliadin peptideP31-43 stimulates expression of inflammatory cytokines in macrophages.

Macrophages in thioglycolate-induced peritoneal exudate cells (PECs)were prepared from B6 WT mice. The macrophages were then stimulated invitro for 3 hours with 100 mg/mL toxic gliadin peptide P31-43, or withthe OVA peptide P323-339 for negative control stimulation.

Expression of the genes for IL-6, IL-15, TNF-α and IFN-β in thestimulated peritoneal exudate cell macrophages was analyzed byquantitative RT-PCR. The expression level of each gene was expressedbased on relative quantification in which the expression level in themacrophages sensitized with the OVA peptide was taken as “1”.

After the stimulation with the gliadin peptide P31-43, the macrophagesof thioglycolate-induced peritoneal exudate cells (PECs) showedsignificantly higher expression of IL-6, IL-15, TNF-β and IFN-α comparedto the macrophages that were sensitized/stimulated with the controlovalbumin P323-339 peptide (FIG. 49).

Example 8Q FIG. 50

CD11b⁺ dendritic cells were isolated from Balb/c WT mice and CD300agene-deficient mice, and then stimulated in vitro for 10 hours with 100mg/mL toxic gliadin peptide P31-43. The expression levels of IL-6,IL-15, TNF-α, IFN-β, MCP1 and MCP5 in the stimulated lamina propriaCD11b+ dendritic cells were analyzed by quantitative RT-PCR. Theexpression level of each gene was expressed based on relativequantification in which the expression level in the WT mice was taken as“1”.

The lamina propria CD11b⁺ dendritic cells of the CD300a gene-deficientmice showed higher levels of expression of the cytokines IL-6, IL-15,TNF-α, IFN-β and MCP1 than the lamina propria CD11b⁺ dendritic cells ofthe WT mice (FIG. 50).

Example 8R FIG. 51

Expression of cytokines and chemokines in lamina propria (LP)macrophages of b6 WT mice and CD300a gene-deficient mice after gliadinstimulation was investigated.

Lamina propria (LP) macrophages derived from b6 WT mice and CD300agene-deficient mice were stimulated in vitro for 3 hours with 100 mg/mLtoxic gliadin peptide P31-43.

Expression of the genes for IL-6, TNF-α and IFN-β in the stimulatedlamina propria (LP) macrophages was analyzed by quantitative RT-PCR. Theexpression level of each gene was expressed based on relativequantification in which the expression level in the WT mice was taken as“1”.

As a result, the lamina propria macrophages of the CD300a gene-deficientmice showed significantly higher expression of IL-6, TNF-α and IFN-βthan those of the WT mice (FIG. 51).

The interaction between CD300a (MAIR-I) of lamina propria macrophagesand phosphatidyl serine on apoptotic cells suppressed the MyD88-mediatedand TRIF-mediated gliadin signaling pathways.

After the in vitro stimulation with the gliadin peptide P31-43, thelamina propria macrophages of the CD300a gene-deficient mice showed highexpression of both inflammatory cytokines and type I IFN (FIG. 43 andFIG. 51).

These results demonstrate that CD300a (MAIR-I) on lamina propriamacrophages in the jejunum suppresses expression of inflammatorycytokines and chemokines enhanced in response to the toxic gliadinpeptide P31-43 derived from dietary gluten.

Example 8S FIG. 52

Expression of cytokines and chemokines in lamina propria (LP)macrophages derived from WT mice or CD300a gene-deficient mice subjectedto depletion of the microbiota (after gliadin stimulation) wasinvestigated.

Lamina propria (LP) macrophages were isolated from antibiotic-treatedBalb/c WT mice and CD300a gene-deficient mice, and stimulated in vitrofor 10 hours with 100 mg/mL toxic gliadin peptide P31-43.

Expression of the genes for IL-6, IL-15, TNF-α, IFN-β, MCP1 and MCP5 inthe stimulated lamina propria (LP) macrophages was analyzed byquantitative RT-PCR. The expression level of each gene was expressedbased on relative quantification in which the expression level in the WTmice was taken as “1”.

High expression of TNF-α and IFN-β in the lamina propria macrophages ofCD300a gene-deficient mice stimulated with gliadin, P31-43, was observednot only in the b6 CD300a gene-deficient mice (FIG. 51), but also in themicrobiota-depleted Balb/c CD300a gene-deficient mice (FIG. 52).

Example 8T FIG. 53

The frequency of phosphatidyl serine-expressing cells in isolated laminapropria (LP) macrophages was investigated.

Lamina propria (LP) macrophages of WT mice and CD300a gene-deficientmice were stained or not stained with annexin V, and subjected todetection of phosphatidyl serine-expressing lamina propria (LP)macrophages in the population gated for CD45⁺PI⁻CD11b⁺CD11c^(low).Annexin V enables detection of changes in the distribution ofphosphatidyl serine in the cell membrane due to apoptosis.

The present inventors demonstrated that CD300a (MAIR-I) is a novelreceptor for phosphatidyl serine (PS), and regulates inflammatoryreaction against bacteria. The isolated lamina propria macrophagescontained annexin V′ cells at less than 10% (FIG. 53).

Example 8U FIG. 54

Macrophages in thioglycolate-induced peritoneal exudate cells (PECs)were prepared from WT mice, and lamina propria (LP) macrophages wereisolated from WT mice and CD300a gene-deficient mice.

Expression of the integrin subunits av and 133 and phosphatidyl serinereceptors in the lamina propria (LP) macrophages of WT mice and CD300agene-deficient mice was investigated.

Example 87 FIG. 55

The expression levels of the genes for αvβ3 integrin andphosphatidylserine receptors (TIM-1, TIM-4, Stabiln-2, SR-PSOX, BAI1 andMER) in these macrophages were analyzed by quantitative RT-PCR. Theexpression level of each gene was expressed based on relativequantification in which the expression level in the macrophages inthioglycolate-induced peritoneal exudate cells of WT mice was taken as“1”.

The lamina propria macrophages and the macrophages inthioglycolate-induced peritoneal exudate cells (PECs) did not showexpression of Tim-1 (FIG. 54).

Tim-4, stabilin-2, SR-PSOX, BAI1 and Mar, which are known as PSreceptors, also did not show significant difference between the laminapropria macrophages of WT mice and the lamina propria macrophages ofCD300a gene-deficient mice (FIG. 54, FIG. 55).

DOCUMENTS

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INDUSTRIAL APPLICABILITY

The present invention provides: an activity modulator that can suppressor promote inhibitory signal transduction of a CD300a-expressing myeloidcell; a medicament for treatment or prophylaxis of a disease or diseasestate in which inhibitory signal transduction of a CD300a-expressingmyeloid cell is involved, which medicament comprises as an effectivecomponent the activity modulator; use of a CD300a gene-deficient mouseas a model mouse; an anti-CD300a antibody having excellent neutralizingaction; and the like.

1-23. (canceled)
 24. An activity modulator for suppressing inhibitorysignal transduction of a CD300a-expressing myeloid cell, said activitymodulator comprising, as a substance that inhibits binding of CD300a tophosphatidyl serine, at least one phosphatidyl serine-binding substanceselected from the group consisting of MFG-E8, MFG-E8 mutants, solubleTIM-1, soluble TIM-4 and soluble stabilin.
 25. An activity modulator forsuppressing inhibitory signal transduction of a CD300a-expressingmyeloid cell, said activity modulator comprising, as a substance thatinhibits binding of CD300a to phosphatidyl serine, an anti-human CD300aantibody comprising: an H-chain variable region having the amino acidsequence of SEQ ID NO:19 or an amino acid sequence that is the same asthe amino acid sequence except that 1, 2, 3, 4, or 5 amino acid(s)is/are substituted, added, inserted and/or deleted; and an L-chainvariable region having the amino acid sequence of SEQ ID NO:20 or anamino acid sequence that is the same as the amino acid sequence exceptthat 1, 2, 3, 4, or 5 amino acid(s) is/are substituted, added, insertedand/or deleted.
 26. An activity modulator for suppressing inhibitorysignal transduction of a CD300a-expressing myeloid cell, said activitymodulator comprising, as a substance that inhibits binding of CD300a tophosphatidyl serine, an anti-mouse CD300a antibody comprising: anH-chain variable region having the amino acid sequence of SEQ ID NO:17or an amino acid sequence that is the same as the amino acid sequenceexcept that 1, 2, 3, 4, or 5 amino acid(s) is/are substituted, added,inserted and/or deleted; and an L-chain variable region having the aminoacid sequence of SEQ ID NO:18 or an amino acid sequence that is the sameas the amino acid sequence except that 1, 2, 3, 4, or 5 amino acid(s)is/are substituted, added, inserted and/or deleted.
 27. An activitymodulator for promoting inhibitory signal transduction of aCD300a-expressing myeloid cell, said activity modulator comprisingphosphatidyl serine as a substance that promotes binding of CD300a tophosphatidyl serine.
 28. A medicament for treatment or prophylaxis of adisease or disease state in which inhibitory signal transduction of aCD300a-expressing myeloid cell is involved, said medicament comprisingas an effective component the activity modulator according to claim 24(excluding MFG-E8 and MFG-E8 mutants).
 29. The medicament according toclaim 28, wherein said disease or disease state in which inhibitorysignal transduction of a CD300a-expressing myeloid cell is involved isan inflammatory infection, allergic disease or autoimmune disease.
 30. Amedicament for treatment or prophylaxis of a disease or disease state inwhich inhibitory signal transduction of a CD300a-expressing myeloid cellis involved, said medicament comprising as an effective component theactivity modulator according to claim
 25. 31. The medicament accordingto claim 30, wherein said disease or disease state in which inhibitorysignal transduction of a CD300a-expressing myeloid cell is involved isan inflammatory infection, allergic disease or autoimmune disease.
 32. Amedicament for treatment or prophylaxis of peritonitis or sepsis causedthereby, said medicament comprising the activity modulator according toclaim 24 (excluding MFG-E8 and MFG-E8 mutants).
 33. A medicament fortreatment or prophylaxis of peritonitis or sepsis caused thereby, saidmedicament comprising the activity modulator according to claim
 25. 34.A medicament for treatment of inflammatory bowel disease, saidmedicament comprising the activity modulator according to claim
 26. 35.A medicament for treatment of celiac disease, said medicament comprisingthe activity modulator according to claim
 27. 36. A medicament fortreatment of atopic dermatitis, said medicament comprising the activitymodulator according to claim
 26. 37. A medicament for treatment ofasthma, said medicament comprising the activity modulator according toclaim
 26. 38. Use of a CD300a gene-deficient mouse for carrying outpathology analysis of an allergic disease or autoimmune disease, or forscreening of a possible candidate substance for an effective componentof a therapeutic agent or prophylactic agent for the disease.
 39. Theuse according to claim 38, comprising use of said CD300a gene-deficientmouse as a model mouse in which inflammatory bowel disease is hardlyinduced after administration of a substance that induces inflammatorybowel disease.
 40. The use according to claim 38, comprising use of saidCD300a gene-deficient mouse as a model mouse that develops celiacdisease after administration of a substance that induces celiac disease.41. The use according to claim 38, comprising the step of: administeringa candidate substance for a therapeutic agent for celiac disease to theCD300a gene-deficient mouse that developed celiac disease, anddetermining the presence or absence of a therapeutic effect; oradministering a candidate substance for a prophylactic agent for celiacdisease together with said substance that induces celiac disease to theCD300a gene-deficient mouse before development of celiac disease, anddetermining the presence or absence of a prophylactic effect.
 42. Theuse according to claim 38, comprising use of said CD300a gene-deficientmouse as a model mouse that hardly develops atopic dermatitis afteradministration of a substance that induces atopic dermatitis.
 43. Theuse according to claim 38, comprising use of said CD300a gene-deficientmouse as a model mouse that hardly develops asthma after administrationof a substance that induces asthma.
 44. An anti-human CD300a antibodycomprising: an H-chain variable region having the amino acid sequence ofSEQ ID NO:19 or an amino acid sequence that is the same as the aminoacid sequence except that 1, 2, 3, 4, or 5 amino acid(s) is/aresubstituted, added, inserted and/or deleted; and an L-chain variableregion having the amino acid sequence of SEQ ID NO:20 or an amino acidsequence that is the same as the amino acid sequence except that 1, 2,3, 4, or 5 amino acid(s) is/are substituted, added, inserted and/ordeleted.
 45. An anti-mouse CD300a antibody comprising: an H-chainvariable region having the amino acid sequence of SEQ ID NO:17 or anamino acid sequence that is the same as the amino acid sequence exceptthat 1, 2, 3, 4, or 5 amino acid(s) is/are substituted, added, insertedand/or deleted; and an L-chain variable region having the amino acidsequence of SEQ ID NO:18 or an amino acid sequence that is the same asthe amino acid sequence except that 1, 2, 3, 4, or 5 amino acid(s)is/are substituted, added, inserted and/or deleted.